A  PRACTICAL  TEXT- BOOK 

OF 

INFECTION,  IMMUNITY 
AND  SPECIFIC  THERAPY 

WITH  SPECIAL  REFERENCE  TO  IMMUNOLOGIC  TECHNIC 


BY 

JOHN  A.  KOLMER,  M.D.,  DR.P.H.,  M.Sc. 

Assistant  Professor  of  Experimental  Pathology,  University  of  Pennsylvania; 
Professor  of  Pathology  and  Bacteriology,  Philadelphia  Polyclinic,  and  Path- 
ologist to  the  Department  of  Dermatologic  Research;  Pathologist  to  the 
Philadelphia  Hospital  for  Contagious  Diseases 


WITH  AN  INTRODUCTION  BY 

ALLEN  J.  SMITH,  M.D.,  Sc.D.,  LL.D. 

Professor  of  Pathology,  University  of  Pennsylvania 


WITH  147  ORIGINAL  ILLUSTRATIONS,  46  IN  COLORS 
By  ERWIN  F.  FABER 

Instructor    of     Medical     Drawing,     University    of     Pennsylvania 


SECOND  EDITION,  THOROUGHLY  RFV'SED 


PHILADELPHIA  AND  LONDON 

W.     B.    SAIJNDERS    COMPANY 

1917 


LIBRARY 


Copyright,  1915,  by  W.  B.  Saunders  Company.     Reprinted  July,  1915. 
Revised,  reprinted,  and  recopyrighted  October,  1917 

Copyright,  1917,  by  W.  B.  Saunders  Company 


PRINTED     IN     AMERICA 

PRESS     OF 

W.     B.    SAUNDERS     COMPANY 
PHILADELPHIA 


TO 
RICHARD   M.   PEARCE,   M.  D.,   D.  SC. 

PROFESSOR  OF  RESEARCH  MEDICINE  IN  THE 
UNIVERSITY    OF    PENNSYLVANIA 


IN    RECOGNITION    OF    HIS    SERVICES   TO   THE 
SCIENCE    OF    MEDICINE 

BY 

THE  AUTHOR 


INTRODUCTION 


THE  last  quarter  of  a  century  has  witnessed  an  almost  marvelous 
development  of  knowledge  in  the  domain  of  medicine  and  the  allied 
sciences,  only  a  part,  of  course,  of  the  extensive  progress  made  in  the  field 
of  general  science.  A  striking  portion  of  this  advance  has  tended  to 
broaden  our  knowledge  of  the  principles  and  of  the  essential  details  of 
the  processes  of  infection  and  immunity,  until  these  branches  have  today 
come  to  form  almost  a  special  science  in  themselves — an  imperium  in 
imperio.  Aside  from  the  personal  factor,  the  writer's  immediate  inter- 
est in  the  present  volume,  as  originally  projected,  arose  from  the  fact 
that  he  was  desirous  of  having  appear  a  series  of  exercises  illustrative  of 
the  principles  of  immunology — a  class-book  intended  to  set  forth  in' 
permanent  form  the  very  excellent  course  of  instruction  that  Dr.  Kolmer 
has  been  giving  during  the  past  few  years  to  selected  groups  of  in- 
terested students  and  occasional  post-graduate  workers  in  the  Medical 
School  of  the  University  of  Pennsylvania.  That  it  should  have  sur- 
passed the  original  simple  plan  and  grown  into  a  volume  of  the  present 
proportions  is  scarcely  to  be  wondered  at/ if  the  temptation  to  elaborate 
the  individual  exercises  by  explanations  and  cognate  considerations  was 
in  the  slightest  to  be  yielded  to.  This  is  due  to  the  fact  that  in  its 
growth  the  subject  has  acquired  so  much  of  undoubted  importance  in 
the  form  of  isolated  observed  facts,  and  itself  presents  so  many  analogies 
and  has  led  to  so  extensive  a  terminology,  that  the  author  who  would 
attempt  to  link  the  observed  facts  into  anything  like  logical  sequence 
or  to  add  in  the  least  to  the  bare  cook-book-like  series  of  illustrative 
exercises  any  explanatory  paragraphs,  cannot  avoid  the  fullness  that 
Dr.  Kolmer  has  found  inevitable  in  presenting  the  subject. 

The  branch  of  immunology,  including  primarily  infection,  and  its 
ramifications  into  diagnosis  and  the  actual  treatment  of  disease,  has 
brought  to  the  parent  subject  of  preventive  medicine  the  greatest  offer- 
ing of  the  decades  of  its  growth.  Itself  contributing  to  world  expansion, 
it  has  nowhere  found  a  greater  stimulus  than  in  the  field  of  exotic  pathol- 
ogy; and  this  last,  in  turn,  has  enriched  internal  medicine,  even  in  its 
most  common  aspects.  The  first  step  in  immunology  may  properly  be 
ascribed  to  Jenner,  with  his  bovine  vaccine  for  smallpox,  a  step  followed 


VI  INTRODUCTION 

only  after  a  long  lapse  of  years  by  Pasteur.  On  the  heels  of  the  latter 
there  appeared  at  once,  and  has  since  then  followed,  an  army  of  men 
whose  names  crowd  the  history  of  the  subject,  and  which  many  of  these 
are  bound  permanently  to  adorn.  The  old  vague  theories  of  infection 
have  taken  form,  and  to  observed  facts  has  been  added  productive 
theory.  The  great  danger  attending  this  luxurious  development  is  that, 
temporarily  at  least,  the  simpler,  and  perhaps  the  more  obvious,  facts 
are  likely  to  be  neglected;  and,  also,  that  symbolization  by  theories 
elaborated  to  harmonize  with  discovered  facts  will  be  accepted  too  fully 
as  explanatory  when  in  reality  it  does  not  explain,  and  that,  as  a  result, 
investigation  will  finally  be  hampered  instead  of  aided.  In  the  almost 
universal  drift  of  experimental  studies  to  internal  stereochemical  factors, 
are  we  not  in  danger  of  placing  too  little  stress  upon  actual  and  possible 
physical  factors?  Is  there  no  danger  that,  by  failing  to  lay  stress  upon 
the  obvious  importance  of  the  turbinate  mechanism  in  the  nose  as  a 
natural  anatomic  factor,  our  rhinologists  may  at  least  feel  justified  in 
sacrificing  this  mechanism  too  readily  for  what  may  be  but  trivial  local 
reasons?  Can  we  insist  that  every  phenomenon  described  with  facility 
in  terms  of  the  side-chain  theory  is  really  a  manifestation  of  chemism, 
when  perhaps,  with  added  investigation  along  lines  of  physical  absorp- 
tion and  the  physical  properties  of  colloids,  an  equally  satisfying  con- 
ception may  be  had,  and  possibly  new  facts  be  developed?  Are  we  not 
blundering  in  rushing  madly  after  matters  of  specificity  as  determined 
by  antigen,  when  perhaps  in  reality  we  are  confronted  by  potential  and 
kinetic  modifications  due  to  peculiarities  of  diet  or  environmental  cir- 
cumstances? The  verity  of  phagocytosis  is  open  to  proof  by  observa- 
tion, and  its  variations  are  likewise  to  be  demonstrated.  Is  the  explana- 
tion of  opsonins  so  convincing  that  merely  the  word  itself  is  enough  to 
satisfy  the  investigator? 

Infection  and  immunity  constitute  a  definite  chapter  in  pathologic 
science.  The  processes  lack  the  dignity  of  a  separate  science  only  in 
that  they  present  variations,  and  the  fact  that  these  are  glossed  over  by 
brilliant  theories  and  conceptions  cannot  prevent  the  deliberate  recogni- 
tion of  serious  incompleteness.  Yet  this  criticism  can  be  applied  to  the 
growth  of  every  branch  of  scientific  knowledge.  It  in  no  wise  militates 
against  the  right  and  the  need  for  setting  forth  the  subject  in  the  light 
that,  for  the  time,  is  afforded  it.  The  importance  of  the  criticism  lies 
only  in  its  acknowledgment,  lest  the  subject  as  at  present  understood 
be  accepted  as  fixed.  With  this  danger  obviated,  and  with  all  theories 
accepted  for  the  time  only  as  working  theories,  and  their  adoption  not 


INTRODUCTION  Vll 

urged  to  curtail  investigations  based  on  other  views,  their  prosecution 
can  be  heartily  applauded. 

This  is  the  view  that  the  writer  believes  that  Dr.  Kolmer  has  had 
in  mind  in  his  presentation  of  the  subject  as  here  set  down.  It  is  cer- 
tainly true  of  the  chapters  that  the  present  writer  has  had  opportunity 
of  examining.  In  such  a  sense,  therefore,  the  work  is  urged  on  the  ap- 
preciation of  the  student,  whether  a  laboratory  worker  or  a  mere  seeker 
of  knowledge. 

I  have  often  been  asked  to  what  extent  I  believe  it  profitable  to  pre- 
sent the  subject  to  the  undergraduate  student.  I  do  not  hesitate  to 
answer  that  so  far  as  the  roster  of  the  medical  curriculum  will  permit, 
the  laboratory  demonstrations  and  exercises  should  form  a  part  of  the 
required  course;  and  that,  with  all  due  caution  to  emphasize  the  fact 
that  our  present  theory  is  not  known  to  be  final,  and  is  offered  merely 
tentatively,  the  verbal  picture  of  the  subject  should  be  outlined  before 
these  beginners.  To  form  some  conception  is  necessary;  and  it  is  better, 
provided  the  mind  be  kept  receptive,  to  follow  a  certain  theory,  even  if 
it  is  unproved,  than  to  do  nothing  at  all  or  to  work  in  confusion.  Our 
American  medical  curriculum  for  undergraduates  is  so  crowded  with 
absolute  essentials  that  the  present  subject  is  habitually  neglected,  save 
for  a  rapid  lecture  outline;  this  is  an  injustice  to  the  student  and  to 
American  medicine.  I  have  tried  to  minimize  this  by  providing, 
through  Dr.  Kolmer's  aid,  a  reasonable  laboratory  course  in  the  essentials 
of  the  branch  to  volunteer  classes  at  first,  at  hours  that  did  not  interfere 
with  the  regular  curriculum — at  present  during  periods  open  to  elec- 
tion. Nevertheless,  the  subject,  influencing  as  it  does  every  branch  of 
medical  practice,  must  take  its  place  with  other  commendable  additions 
to  the  required  schedule.  That  this  can  be  done  only  by  lengthening 
the  course  of  study,  either  in  the  annual  session  or  by  adding  a  year  to 
our  present  four-year  course,  is  obvious,  and  to  that  end  we  are  rapidly 
approaching. 

ALLEN  J.  SMITH. 


PREFACE  TO  THE  SECOND  EDITION 


THE  purpose  and  general  plan  of  this  volume  remains  unchanged 
in  this  edition.  I  have  endeavored  to  make  it  a  practical  treatise  for 
medical  students,  practitioners,  and  laboratory  workers  by  setting 
forth  theories  and  opinions  simply  and  plainly,  and  devoting  particular 
attention  to  technic  and  the  practical  phases  and  application  of  the 
subjects  considered;  I  hope  that  this  edition  will  receive  the  same  gen- 
erous recognition  as  its  predecessor. 

Additions  and  alterations  have  been  made  throughout;  special 
attention  has  been  given  the  subject  of  focal  infection;  the  Schick  toxin 
test  for  immunity  in  diphtheria  and  active  immunization  in  diphtheria 
with  toxin-antitoxin  mixtures;  complement-fixation  in  tuberculosis  and 
other  bacterial  infections  and  a  quantitative  Wassermann  reaction 
based  upon  my  studies  with  the  co-operation  and  assistance  of  Dr. 
Claude  P.  Brown,  Dr.  Toitsu  Matsunami,  and  Dr.  Berta  Meine,  aiming 
to  standardize  this  important  test.  The  chapters  on  anaphylaxis  have 
been  revised  and  particular  attention  given  the  subject  of  anaphylactic 
skin  reactions.  Lange's  colloidal  gold  reaction  has  been  included. 
The  chapter  on  the  treatment  of  various  infections  with  bacterial  vac- 
cines has  been  enlarged  and  the  non-specific  activity  of  bacterial  vac- 
cines discussed.  The  section  on  the  treatment  of  certain  of  the  acute 
infectious  diseases,  and  particularly  acute  anterior  poliomyelitis,  with 
the  serum  of  convalescents  and  normal  persons  has  been  amplified; 
blood  transfusion  has  been  included.  Special  attention  has  been  de- 
voted to  the  chapter  on  Chemotherapy,  and  the  results  of  the  studies  of 
Dr.  Jay  F.  Schamberg,  Dr.  George  Raiziss,  and  the  author  bearing  upon 
the  toxicity  of  salvarsan  and  its  congeners  and  the  reactions  following 
their  administration  have  been  included  and  discussed.  The  subject 
of  Bacterial  Chemotherapy,  which  promises  much  in  the  future,  has  been 
amplified  from  the  theoretical  and  technical  viewpoints. 

The  section  on  Experimental  Infection  and  Immunity  remains  as 
part  of  the  volume  instead  of  forming  a  separate  book;  experience  has 
shown  that  this  is  a  good  plan,  and  enables  the  student  doing  more  or 
less  independent  work  to  consult  the  text  for  directions  and  discussions. 

J.  A.  K. 

MCMANES  LABORATORY  OF  EXPERIMENTAL  PATHOLOGY, 
UNIVERSITY  OF  PENNSYLVANIA,  October,  1917. 

ix 


PREFACE 


FOR  the  past  twenty  years  the  science  of  immunity  has  been  one  of 
the  most  progressive  and  most  active  branches  in  the  department  of 
medicine.  An  enormous  literature  has  accumulated;  many  new  terms 
have  been  coined,  and  numerous  theories  have  been  adduced;  indeed, 
the  subject  has  acquired  an  aspect  of  complexity  that  is  confusing  to 
those  not  specially  interested  or  engaged  in  this  work. 

The  purpose  of  this  book  is  a  threefold  one,  namely : 

1 .  To  give  to  practitioners  and  students  of  medicine  a  connected  and  con- 
cise account  of  our  present  knowledge  regarding  the  manner  in  which  the 
body  may  become  infected,  and  the.  method,  in  turn,  by  which  the  organism 
serves  to  protect  itself  against  infection,  or  strives  to  overcome  the  infection 
if  it  should  occur,    and  also  to  present   a   practical  application  of  this 
knowledge  to  the  diagnosis,  prevention,  and  treatment  of  disease. 

2.  To  give  to  physicians  engaged  in  laboratory  work  and  special  workers 
in  this  field  a  book  to  serve  as  a  guide  to  the  various  immunologic  methods. 

3.  To  outline  a  laboratory  course  in  experimental  infection  and  im- 
munity for  students  of  medicine  and  those  especially  interested  in  these 
branches. 

1.  The  subject  of  infection  is  intimately  connected  with  that  of 
immunity,  and  this  is  especially  emphasized  in  those  diseases  for  which 
a  specific  therapy  exists,  for  a  knowledge  of  the  nature  of  the  infection 
is  of  paramount  importance  in  controlling  the  dosage  and  indicating  the 
method  of  administration  of  a  specific  therapeutic  agent.  By  describing 
principles  and  technic  with  considerable  detail,  a  special  effort  has  been 
made  to  render  Part  IV  of  this  book  of  particular  value  to  practitioners 
of  medicine. 

The  day  is  past  when  the  physician  and  surgeon  can  relegate  the  things 
of  immunity  entirely  to  the  laboratory.  Diagnostic  methods  and  re- 
actions and  the  field  of  specific  therapy, — vaccine,  serum,  and  chemo-, — 
are  subjects  of  such  practical  importance  that  it  is  obvious  that  the 
physician  and  the  student  of  medicine  can  no  longer  be  merely  mildly 
interested  onlookers.  The  physician  who  injects  salvarsan,  a  serum, 
or  a  vaccine,  or  who  uses  a  diagnostic  reaction,  must  be  prepared  to  ex- 
plain to  his  patient  the  nature  of  the  therapy  he  employs  and  the  sig- 


Xll  PREFACE 

nificance  of  the  reaction.  This  he  can  do  only  by  equipping  himself 
with  the  knowledge  of  the  fundamental  factors  of  immunity,  or  he  will 
be  forced  into  the  position  of  a  passive  transmitter  of  ideas  entirely  be- 
yond his  own  knowledge. 

2.  An  effort  has  been  made  to  include  data  of  both  practical  and 
theoretic  importance,  and  in  some  instances  tests  are  described  that  are 
more  of  theoretic  than  of  practical  import,  especially  in  research  work. 

»  It  is  obviously  impossible,  in  a  single  volume,  to  include  the  very 
large  number  of  tests  and  modifications  that  have  been  advocated  from 
time  to  time,  and,  as  a  matter  of  course,  most  attention  has  been  given 
those  methods  that  have  been  shown  to  be  of  practical  value  or  that 
give  promise  of  becoming  so.  So  far  as  possible  original  methods  are 
given,  these  being,  in  the  larger  proportion,  more  or  less  important  modi- 
fications devised  as  the  result  of  my  own  experience  in  hospital  and  teach- 
ing laboratories. 

The  technic  of  the  various  tests  and  reactions  is  described  in  great 
detail,  thus  tending  the  better  to  secure  accuracy,  simplicity,  and  definite- 
ness,  and  to  serve  as  an  opening  wedge  to  those  about  to  enter  this 
special  field. 

3.  The  value  of  the  experimental  method  in  the  teaching  of  certain 
branches  of  medicine  is  now  well  recognized.     In  no  department,  how- 
ever, is  this  method  of  greater  value  than  in  the  study  of  infection  and 
immunity.     A  working  knowledge  of  these  subjects  is  so  valuable  in  the 
practice  of  medicine  and  surgery  that  the  student  should  be  well  versed 
in  at  least  their  primary  principles  and  practical  applications  in  the 
prophylaxis,  diagnosis,  and  treatment  of  diseas 

The  laboratory  course  given  in  Part  V  is  based  upon  the  courses 
given  by  me  in  the  Laboratory  of  Experimental  Pathology  at  the  Uni- 
versity of  Pennsylvania,  and  in  the  laboratories  of  the  Philadelphia 
Polyclinic  and  College  for  Graduates  in  Medicine.  In  including  them 
in  this  volume  I  am  carrying  out  my  original  plan,  for  in  many  of  the 
experiments  the  exact  technic  of  a  given  test  is  described,  making  a  sepa- 
rate book  devoted  to  this  part  of  the  subject  unnecessary.  Future  ex- 
perience may,  however,  show  the  necessity  of  having  this  portion  of  the 
book  form  a  separate  laboratory  manual.  I  shall  appreciate  the  opin- 
ions of  educators  who  may  have  occasion  to  consult  the  course  herein 
outlined. 

Since  the  larger  portion  of  our  knowledge  of  infection  and  immunity 
has  been  gained  from  studies  upon  the  lower  animals,  it  is  not  strange 
that  these  were  early  and  directly  benefited  by  a  practical  application 


PREFACE  Xlii 

of  this  knowledge  to  the  prophylaxis,  diagnosis,  and  treatment  of  many 
of  the  diseases  to  which  these  animals  are  subject.  I  have,  therefore, 
included  in  this  volume  an  account  of  those  immunologic  diagnostic 
reactions  and  applications  of  specific  therapy  that  have  a  direct  bearing 
upon  veterinary  medicine. 

No  attempt  has  been  made  to  cover  all  literature  references  on  the 
subject.  An  effort  has  been  made  to  state  well-established  facts  con- 
cisely, and,  in  the  case  of  the  more  recent  subjects,  to  give  the  principal 
references  to  the  literature.  I  have  drawn  largely  from  German, 
French,  and  English  sources,  and  have  endeavored,  wherever  possible, 
to  give  proper  and  due  credit  to  each  author.  In  order  to  keep  the  work 
up  to  the  times,  I  would  ask  the  authors  of  reprints  on  immunologic 
subjects  to  send  me  copies. 

The  illustrations,  all  of  which  have  been  made  by  Mr.  Erwin  F. 
Faber,  will,  it  is  hoped,  serve  the  purpose  for  which  they  are  intended, 
namely,  to  elucidate  the  text  and  to  teach,  rather  than  merely  to  em- 
bellish. 

It  is  with  deep  and  sincere  appreciation  that  I  acknowledge  the  en- 
couragement and  aid  given  me  by  Professor  Allen  J.  Smith,  who  has 
written  the  introduction  and  reviewed  several  chapters.  My  thanks 
are  also  due  to  Professor  Richard  M.  Pearce  for  reviewing  the  chapters 
on  Infection;  to  my  assistant,  Dr.  Anna  M.  Raiziss,  for  a  number  of 
translations  from  foreign  literature,  and  to  the  publishers,  whose  kind 
and  unvarying  courtesy  has  greatly  simplified  the  work. 

J.  A.  K. 

MCMANES  LABORATORY  OF  EXPERIMENTAL  PATHOLOGY, 
UNIVERSITY  OF  PENNSYLVANIA. 


CONTENTS 


PAGE 
INTRODUCTION v 

PART    I 

GENERAL  IMMUNOLOGIC  TECHNIC 

CHAPTER  I. — GENERAL  TECHNIC 17 

Care  of  centrifuge,  17 — Making  a  simple  capillary  pipet,  18 — Making 
looped  pipets,  20 — Graduated  pipets,  21 — Making  Wright  blood-capsules, -23 — 
Making  vaccine  ampules,  24 — Preparation  of  test-tubes  for  immunologic 
work,  25 — Selection  of  a  satisfactory  syringe,  26 — Solutions,  27. 

CHAPTER  II. — METHODS  OF  OBTAINING  HUMAN  AND  ANIMAL  BLOOD 28 

Obtaining  corpuscles,  28 — Washing  erythrpcytes,  28 — Obtaining  serum,  30 
— Obtaining  corpuscles  and  serum,  30 — Obtaining  blood  plasma,  30— Obtaining 
small  amounts  of  human  blood,  32 — Obtaining  larger  amounts  of  human  blood 
(phlebotomy,  wet-cupping,  placental  blood),  33 — Obtaining  cerebrospinal  fluid 
(technic  of  spinal  puncture),  37 — Obtaining  small  amounts  of  animal  blood 
(rabbit,  guinea-pig,  sheep),  41 — Obtaining  large  amounts  of  animal  blood  (rab- 
bit, guinea-pig,  rat,  sheep,  hog,  monkey,  dog,  and  horse),  42. 

CHAPTER  III. — TECHNIC  OF  ANIMAL  INOCULATION 53 

General  rules,  53 — Method  of  subcutaneous  inoculation  (fluid  and  solid 
inocula),  54 — Method  of  intramuscular  inoculation,  56 — Methods  of  intravenous 
inoculation  (rabbit,  guinea-pig,  mice  and  rats,  horse,  sheep,  goat,  dog),  56 — 
Method  of  intracardial  inoculation,  62 — Methods  of  intraperitoneal  inoculation 
(rabbit,  guinea-pig),  64. 

CHAPTER  IV. — METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF  ANIMALS  . .     65 

Antigens  and  active  immunization,  65 — General  technic,  66 — Production  of 
antitoxins,  68 — Production  of  agglutinins  (intravenous  and  intraperitoneal  inocu- 
lation), 68 — Production  of  immune  opsonins,  69 — Production  of  bacteriolysins, 
69 — Production  of  precipitins,  70 — Production  of  hemolysins  (intravenous  and 
intraperitoneal  inoculations),  71 — Production  of  cytotoxins,  73. 

CHAPTER  V. — PRESERVATION  OF  SERUMS — METHODS 75 

Methods  for  the  preservation  of  normal  serums,  75 — Methods  for  the  preser- 
vation of  immune  serum  in  fluid  form  with  antiseptics,  76 — In  fluid  form  by  bac- 
teria-free filtrations,  76 — In  fluid  form  by  freezing,  79 — Preservation  in  powder 
form,  79 — Preservation  in  dried  paper  form,  80. 

PART   II 

PRINCIPLES  OF  INFECTION 

CHAPTER  VI.  —INFECTION 81 

Definition,  81 — Relation  of  infection  to  immunity,  83 — Source  of  infection, 
83 — Contagious  and  infectious  diseases,  84 — Exogenous  and  endogenous  in- 
fection, 85— -Avenues  of  infection,  86 — Normal  defenses  against  bacterial  in- 
vasion, 90 — Mechanism  of  bacterial  invasion,  91 — Local  infection,  94 — Mechan- 
ism of  infection,  94 — The  avenue  of  infection  and  tissue  susceptibility,  98 — 
The  numeric  relationship  of  bacteria  to  infection,  99 — General  susceptibility 
in  relation  to  infection,  100 — The  defensive  mechanism  of  the  microorganism 
in  relation  to  infection,  103 — Mixed  infection,  106 — Summary,  107. 


4  CONTENTS 

PAGE 

CHAPTER  VII. — INFECTION  (continued).     PRODUCTION  OF  DISEASE 108 

Toxins,  109 — Extracellular  bacterial  toxins,  110 — General  properties  of 
soluble  toxins,  110— Precipitation  of  the  extracellular  toxins,  111 — Struc- 
ture of  soluble  toxins,  111 — Nature  of  soluble  toxins,  112 — Selective  action 
of  soluble  toxins,  112.  Special  Properties  of  the  Principal  Soluble  Toxins, 
113 — Diphtheria  toxin,  113 — The  guinea-pig  test  for  virulence  of  diphtheria 
bacilli,  114 — Tetanus  toxin,  117 — Botulism  toxin,  117 — Dysentery  toxin,  118 
— Staphylotoxin,  118— Streptotoxin,  119.  Toxins  of  the  Higher  Plants  and 
Animals,  119 — Phytotoxins,  119 — Pollen  toxin,  120— Zootoxins,  121 — Snake 
venom,  121.  Endotoxins,  122 — Methods  of  studying  endotoxins,  122 — 
Nature  of  endotoxins,  123 — Aggressins,  124 — Bail's  classification  of  bacteria, 
125 — Nature  of  aggressins,  126 — Anti-aggressins,  127 — .Bacterial  Proteins,  127 — 
Bacterial  split  protein,  128 — Nature  of  bacterial  proteins,  128 — Action  of  bac- 
terial proteins,  129 — Theory  of  Vaughan,  130.  Ptomains,  131.  Mechanical 
Action  of  Bacteria,  133 — Infection  with  Animal  Parasites,  134 — The  Course  of 
Infection,  136 — Stages  of  infection,  136 — Grades  of  Infection,  138 — Systemic 
reaction  to  infection,  139. 

PART   III 

PRINCIPLES  OF  IMMUNITY  AND  SPECIAL  IMMUNOLOGIC 

TECHNIC 

CHAPTER  VIII. — IMMUNITY. — THEORIES  OF  IMMUNITY 140' 

Definition,  142 — Historic,  142 — Exhaustion  theory  of  immunity,  143 — 
Retention  theory  of  immunity,  146 — Theory  of  phagocytosis,  146 — Side-chain 
theory  of  immunity,  148 — Compatibility  of  the  phagocytic  and  side-chain 
theories,  157 — Antigens,  161 — Antibodies,  163. 

CHAPTER  IX. — VARIOUS  TYPES  OF  IMMUNITY 167 

Natural  Immunity,  167 — Explanation  of  natural  immunity,  168 — Non- 
specific immunity,  168 — Local  immunity,  169 — Phagocytosis  and  natural  im- 
munity, 170— Natural  antitoxic  immunity,  171 — Natural  bacteriolytic  immun- 
ity, 171 — Natural  anti-aggressin  immunity,  171 — Athreptic  immunity,  172. 
Acquired  Immunity,  172 — Active  acquired  immunity,  172 — Passive  acquired  im- 
munity, 174. 

CHAPTER  X. — PHAGOCYTOSIS 177 

Historic,  177 — The  original  theory  of  phagocytosis,  178— Kinds  of  phagocy- 
tosis, 179 — The  relation  of  the  cell  types  to  infection,  180 — Chemotaxis,  181 — 
Positive  chemotaxis,  181 — Negative  chemotaxis,  184 — Results  of  phagocytosis, 
184 — The  relation  of  body  fluids  to  phagocytosis,  186 — Revised  theory  and  role 
of  phagocytosis  in  immunity,  188 — Mechanism  of  phagocytosis,  189. 

CHAPTER  XI. — OPSONINS 190 

Historic,  190 — Definition,  191— Properties  and  nature  of  opsonins,  191— 
Susceptibility  to  opsonification,  192— Effect  of  opsonins  on  bacteria,  192— Role 
of  opsonins  in  immunity,  193. 

CHAPTER  XII. — OPSONIC  INDEX 194 

Principles  involved,  194 — Definition,  194 — Purpose  of  the  opsonic  index, 
195— Limitations  of  the  method,  195 — Precautions  in  technic,  196— Technic  of 
the  opsonic  index  (Wright),  196 — Technic  of  quantitative  estimation  of  bacterio- 
tropins  in  immune  serum  (Neufeld),  203 — Practical  value  of  the  opsonic  index, 
206 — Value  of  the  index  in  diagnosis,  207 — In  prognosis,  208 — As  a  guide  to 
bacterial  vaccine  therapy,  208. 

CHAPTER  XIII. — BACTERIAL  VACCINES 210 

Definition,  210 — Technic  of  preparing  bacterial  vaccines,  210— Counting 
a  bacterial  vaccine  (method  of  Wright),  214 — Counting  with  the  hemocytometer 
chamber,  215— Method  of  Kolle,  216— Method  of  Hopkins,  216— Preparation  of 
"sensitized"  bacterial  vaccine,  221 — The  administration  of  a  bacterial  vaccine, 
221 — Making  the  inoculation,  221 — Effects  of  inoculation,  222— Frequency  and 
dosage  of  inoculation,  222 — Ordinary  adult  doses  of  the  common  vaccines,  222. 


CONTENTS  5 

PAGE 

CHAPTER  XIV. — ANTITOXINS 224 

Definition,  224 — Historic,  224 — Formation  of  antitoxins,  225 — Structure  of 
antitoxins,  227 — Properties  of  antitoxins,  227 — Natural  antitoxins,  228 — Schick 
test  for  natural  diphtheria  antitoxin,  228 — Specificity  of  antitoxins,  232 — 
Nature  of  the  toxin-antitoxin  reaction,  232 — Production  of  Diphtheria  Anti- 
toxin, 235 — Production  of  diphtheria  toxin,  235 — Testing  the  toxin,  236 — Im- 
munizing the  animals,  236 — Collecting  the  serum,  239 — Method  of  concentrat- 
ing, 239— Standardizing  the  serum,  240.  Production  of  Tetanus  Antitoxin,  243 
—Tetanus  toxin,  243 — Immunizing  the  animals,  244 — Collecting  the  serum,  244 
— Standardizing  the  serum,  244.  Botulimis  Antitoxin,  245.  Antidysentery 
Serum,  245 — The  culture,  246 — Immunizing  the  animals,  247 — Rapid  produc- 
tion, 247 — Collecting  and  testing  the  serum,  248.  Antistaphylococcus  Serum, 
249 — Preparation,  249 — Technic  of  the  anti-lysin  test,  249.  Production  of 
Antivenin,  251.  Production  of  Pollen  Antitoxin,  252.  The  Measure  of  Anti- 
toxins, 252 — A  unit,  252 — Unit  of  diphtheria  antitoxin,  253 — Unit  of  tetanus 
antitoxin,  253. 

CHAPTER  XV. — FERMENTS  AND  ANTIFERMENTS 254 

Bacterial  ferments,  254 — Similarity  between  toxins  and  ferments,  254 — 
Antif erments,  256 — Antibodies  and  antiferments,  257 — Antiferments  in  disease, 
257 — Ferments  in  pregnancy  and  disease,  258.  Mechanism  of  the  Abderhalden 
reaction,  260.  Ferment  Reactions,  264— Antitrypsin  test,  264— Abderhalden 's 
serodiagnosis  of  pregnancy,  265 — Practical  value  of  Abderhalden's  test,  276 
— Sero-enzymes  in  cancer,  277 — In  mental  diseases,  278 — In  syphilis,  278 — In 
tuberculosis  and  acute  infection,  278. 

CHAPTER  XVI. — AGGLUTININS 279 

Definition,  279 — Historic,  279— Normal  and  immune  agglutinins,  281 — 
Formation  of  agglutinins,  281 — Origin  of  agglutinins,  282 — Properties  and 
nature  of  agglutinins,  283 — Acid  agglutination,  283 — Mechanism  of  agglutina- 
tion, 284 — Specificity  of  agglutinins,  285 — Absorption  methods  for  differentiat- 
ing between  a  mixed  and  single  infection,  285 — Hemagglutinins,  286 — Non-ag- 
glutinable  species  of  bacteria,  288 — Variation  in  agglutinating  strength  of  a 
serum,  288 — Conglutination,  289 — Role  of  agglutinins  in  immunity,  289. 
Practical  Applications,  290 — In  the  diagnosis  of  typhoid  fever,  290 — Paraty- 
phoid fever,  291 — Dysentery,  292 — Cholera,  292— Cerebrospinal  meningitis, 
292— Plague,  292— Malta  fever,  292— Pneumonia,  292— Syphilis,  292— Per- 
tussis, 293 — Glanders,  293 — In  the  differentiation  of  bacteria,  293 — In  the  diag- 
nosis of  single  and  mixed  infection,  294.  The  Agglutination  Reaction,  294— 
Microscopic  method  with  serum,  290 — Microscopic  method  with  dried  blood, 
300 — Macroscopic  method,  301 — Differentiation  of  pneumococci,  308 — The 
saturation  test  of  Castellani,  309 — Technic  of  acid  agglutination,  310.  Tests 
before  Blood  Transfusion  for  Isohemagglutinins  and  Isohemolysins,  311 — 
Microscopic  method  of  Brem,  312. 

CHAPTER  XVII. — PRECIPITINS 314 

Definition,  314 — Historic,  314 — Nomenclature,  316 — Structure  and  propor- 
tion of  precipitins,  316 — Formation  of  precipitins,  317 — Mechanism  of  precipita- 
tion, 318 — Specificity  of  precipitins,  318 — Role  of  precipitins  in  immunity,  319. 
Practical  Applications,  320 — Bacterial  precipitins,  320 — Fornet  ring  test,  321 — 
Porges-Meier  reaction,  321 — Herman-Perutz  reaction,  322— Noguchi  globulin  re- 
action, 322 — McDonagh  ''gel"  test,  323 — Differentiation  of  proteins,  324. 
Technic  of  the  Precipitin  Reactions,  326 — Differentiation  of  human  and  animal 
bloods,  326 — Detection  of  meat  adulteration,  333 — Bacterial  precipitins,  336. 

CHAPTER  XVIII. — CYTOLYSINS.     AMBOCEPTORS  AND  COMPLEMENTS 338 

Definition,  339 — Kinds  of  cytolysins,  340 — Nomenclature,  340.  Ambocep- 
tors,  341 — Historic,  341 — Structure  of  amboceptors,  342 — General  properties  of 
amboceptors,  343 — Mechanism  of  the  action  of  amboceptors,  343 — Formation 
of  amboceptors,  344 — Quantitative  estimation  of  amboceptors,  347 — Titration 
of  hemolytic  amboceptor,  347 — Titration  of  bacteriolytic  amboceptor,  347. 
Complements,  348 — Historic,  348 — Definition,  348 — Structure  and  general 
properties  of  complement,  348 — Anticomplements,  349— Origin  of  complements, 
350 — Multiplicity  of  complements,  350 — Endocomplements,  352 — Complement- 
splitting,  353 — Complement  fixation,  354 — Complement  deviation,  355 — Quan- 
titative titration  of  complement,  356. 


D  CONTENTS 

CHAPTER  XIX. — BACTERIOLYSINS 358 

Historic,  358 — Definition,  359 — Origin  of  bacteriolysins,  360 — Leukins  and 
leukocytic  extracts,  360 — Method  of  preparing  leukocytic  extracts,  361 — Mech- 
anism of  bacteriolysis,  362 — General  properties  of  bacteriolysins,  363 — Normal 
bacteriolysins,  363 — Specificity  of  bacteriolysins,  363.  Practical  Applications, 
363— Technic  of  the  Pfeiffer  test,  364 — Bacteriolytic  test  in  vivo  f9r  the  identi- 
fication of  bacteria,  365 — Bacteriolytic  test  in  vivo  in  the  diagnosis  of  disease, 
370 — Bacteriolytic  test  in  vitro  (method  of  Stem  and  Korte),  371 — Bacterio- 
lytic test  in  vitro  (method  of  Wright),  377. 

CHAPTER  XX. — HEMOLYSINS 383 

Historic,  383 — Definition,  384 — Nomenclature,  385 — Nature  of  hemolysins, 
385 — Analogy  between  bacteriolysis  and  hemolysis,  389 — Specificity  of  hemoly- 
sins, 390 — Normal  hemolysins,  390 — Production  of  immune  hemolysins,  393 — 
General  properties  of  hemolysins,  393 — Source  of  hemolysins,  394.  Practical 
Applications,  395 — Quantitative  reactions  between  hemolytic  amboceptor  and 
complement,  396 — Method  of  titration  of  hemolysin,  397 — Method  for  removing 
hemolysin  from  a  serum,  400 — Method  of  determining  natural  hemolysins  in 
serum,  400-^The  serum  diagnosis  of  paroxysmal  hemoglobinuria,  401 — Method 
of  determining  the  resistance  of  red  blood-corpuscles,  402. 

CHAPTER  XXI. — VENOM  HEMOLYSIS : 405 

Historic — nature  of  venom  hemolysis,  405— Venom  hemolysis  in  syphilis, 
407 — Practical  value  of  the  venom  test  in  syphilis,  410 — The  psycho-reaction  of 
Much  in  syphilis,  410 — Venom  hemolysis  in  tuberculosis,  412 — Venom  hemolysis 
in  cancer,  412. 

CHAPTER  XXII. — PRINCIPLES  OF  THE  PHENOMENON  OF  COMPLEMENT  FIXATION  413 

Historic,  413 — The  original  complement-fixation  method  of  Bordet,  416 — 
Mechanism  of  complement  fixation,  417 — Non-specific  complement  fixation, 
418 — Quantitative  factors  in  complement-fixation  tests,  421 — Practical  applica- 
tions, 423. 

CHAPTER  XXIII. — THE  TECHNIC  OF  COMPLEMENT-FIXATION  REACTIONS 425 

The  Wassermann  Syphilis  Reaction,  425 — Historic,  425 — Principles  and 
theories  of  the  syphilis  reaction,  428 — General  technic,  431 — Preparation  of  the 
fluid  to  be  tested,  432 — Preparation  and  titration  of  complement,  435— Preserva- 
tion of  complement,  437 — Preparation  and  titration  of  hemolytic  amboceptor, 
439 — Preparation  of  blood-corpuscles,  440 — Preparation  and  standardization  of 
antigens,  441— Technic  of  the  First  Method,  459— Technic  of  the  Second  Method, 
465-^Technic  of  the  Third  Method,  467 — Technic  of  the  Fourth  Method,  470. 
Modifications  of  the  Wassermann  Reaction,  475 — Technic  of  the  Noguchi  modi- 
fication, 476 — Technic  of  the  Hecht-Gradwohl  modification,  482 — Other  modi- 
fications, 484.  The  Wassermann  Reactions  in  the  Various  Stages  of  Syphilis, 
485 — The  specificity  of  the  Wassermann  reaction,  490 — The  effect  of  treatment 
upon  the  Wassermann  reaction,  492 — The  Practical  Value  of  the  Wassermann 
reaction,  495. 

CHAPTER  XXIV. — COMPLEMENT-FIXATION  REACTIONS  (continued) 498 

Specific  complement  fixation  in  bacterial  diseases,  498 — Preparation  of 
bacterial  antigens,  498 — Standardizing  bacterial  antigens,  500 — Principles  of 
complement  fixation  with  bacterial  antigens,  501.  Complement  Fixation  in  the 
Differentiation  of  Microparasites,  502.  Complement  Fixation  in  Gonococcus 
Infections,  503.  Complement  Fixation  in  Glanders,  511.  Complement  Fixa- 
tion in  Contagious  Abortion,  513.  Complement  Fixation  in  Dourine,  515. 
Complement  Fixation  in  Typhoid  Fever,  516.  Complement  Fixation  in  Tuber- 
culosis, 517.  Complement  Fixation  in  Pertussis,  521.  Complement  Fixation 
in  the  Standardization  of  Immune  Serums,  524.  Complement  Fixation  in 
Echinococcus  Disease,  524.  Complement  Fixation  in  the  Differentiation  of 
Proteins  (blood-stains,  meats,  bacteria),  527.  Complement  Fixation  in  Cancer, 
532. 


CONTENTS  7 

PAQK 

CHAPTER  XXV.— CYTOTOXINS 534 

Nomenclature,  534 — Nature  and  general  properties  of  cytotoxins,  534 — 
Preparation  of  cytotoxins,  535 — Methods  of  studying  cytotoxins,  535 — Specific- 
ity of  cytotoxins,  536 — Autocytotoxins,  537 — Isocytotoxins,  538 — Anticytotoxic 
serums,  538 — Kinds  of  cytotoxins,  538 — Spermatotoxin,  538 — Epitheliotoxin, 
538 — Leukotoxin,  538 — Nephrotoxin,  538 — Hepatotoxin,  539 — Gastrotoxin, 
539 — Syncytotoxin,  539 — Neurotoxin,  540 — Thyrotoxin,  540 — Role  of  cyto- 
toxins in  immunity,  540 — Practical  applications,  540 — Cytotoxic  cancer  reac- 
tion, 541. 

CHAPTER  XXVI. — THE  RELATION  OF  COLLOIDS  AND  LIPOIDS  TO  IMMUNITY  . . .  543 

Kinds  of  colloids,  543 — Nature  and  properties  of  colloids,  543 — Analogy  be- 
tween the  reactions  of  immunity  and  those  of  colloidal  chemistry,  548 — Anti- 
toxins, 548 — Agglutinins  and  precipitins,  55fr— Hemolysins,  551 — Complement 
fixation  as  a  colloid  reaction,  552 — The  relation  of  lipoids  to  immunity,  553 — 
Lange's  colloidal  gold  reaction,  555 — The  epiphanin  reaction,  559 — The  miostag- 
min  reaction,  563. 

CHAPTER  XXVII. — ANAPHYLAXIS 567 

Historic,  568 — Definition,  571 — Phenomena  of  anaphylaxis,  572 — Mechan- 
ism of  anaphylaxis,  577 — Anaphylactogens  or  allergens,  579 — The  humeral  or 
anaphylotoxin  theories  of  anaphylaxis,  584 — Anaphylactin  (allergin),  588 — 
Theories  of  anaphylaxis,  591 — Cellular  theory  of  anaphylaxis,  594r— Passive 
anaphylaxis,  596 — Anti-anaphylaxis  or  desensitization,  599 — Specificity  of 
anaphylaxis,  601. 

PART   IV 

APPLIED  IMMUNITY  IN  THE  PROPHYLAXIS,  DIAGNOSIS,  AND 
TREATMENT  OF   DISEASE— SPECIFIC   THERAPY 

CHAPTER  XXVIII. — ANAPHYLAXIS  IN  ITS  RELATION  TO  INFECTION  AND  IM- 
MUNITY.   ANAPHYLACTIC  OR  ALLERGIC  REACTIONS 602 

Relation  of  anaphylaxis  to  infectious  diseases,  603 — Relation  of  anaphylaxis 
to  non-infectious  diseases,  608 — Serum  disease,  608 — Idiosyncrasies,  614 — 
Relation  of  anaphylaxis  to  immunity,  618 — Anaphylactic  or  allergic  skin  reac- 
tions, 620 — Subcutaneous  tuberculin  reaction,  633 — Intracutaneous  tuberculin 
reaction,  636 — Cutaneous  tuberculin  reaction,  637 — Conjunctival  tuberculin  re- 
action, 639 — Percutaneous  tuberculin  reaction,  640— Tuberculin  reactions 
among  the  lower  animals,  641 — The  luetin  reaction,  642 — The  mallein  reaction, 
647 — Abortin  reaction,  648— Allergic  reactions  in  typhoid  fever,  649 — Allergic 
reactions  in  other  diseases,  652. 

CHAPTER  XXIX.— ACTIVE  IMMUNIZATION.     VACCINES  IN  THE  PROPHYLAXIS 

AND  TREATMENT  OF  DISEASE.     VACCINE  THERAPY 654 

Historic,  654 — Nomenclature,  656 — Method  of  preparing  vaccines,  657 — 
Mechanism  of  active  immunization,  659 — Non-specific  activity,  663 — Living 
versus  dead  vaccines,  664 — Sensitized  vaccines,  665 — Autogenous  versus  stock 
bacterial  vaccines,  665 — The  negative  phase,  666 — Contraindications  to  active 
immunization,  667.  Prophylactic  Irtimunization  or  Vaccination,  668 — In  small- 

?ox,  668 — In  rabies,  681 — In  typhoid  fever,  688 — In  paratyphoid  fever,  693 — 
n  typhus  fever,  694 — In  pneumonia,  694 — In  plague,  694 — In  cholera,  697 — In 
dysentery,  699 — In  cerebrospinal  meningitis,  699 — In  scarlet  fever,  699 — In 
pertussis,  700 — In  anthrax,  700 — In  black-leg,  702.  Therapeutic  Immunization, 
Bacterial  Vaccine  Therapy,  702— Principles,  702— Infected  wounds,  703 — Focal 
infections,  704 — Diseases  of  the  skin,  704 — Diseases  of  the  genito-urinary  sys- 
tem, 706 — Diseases  of  the  respiratory  system,  708 — Acute  general  infections, 
713—  Tuberculin  therapy,  713. 


QE 

i 


8  CONTENTS 

PAGE 
CHAPTER  XXX. — PASSIVE  IMMUNIZATION.     SERUMS  IN  THE  PROPHYLAXIS  AND 

TREATMENT  OF  DISEASE.     SERUM  THERAPY 73 

Definition,  733— Purposes  of  passive  immunization,  734 — Kinds  of  passive 
immunity,  735 — Indications  for  passive  immunization,  736 — Contraindications 
to  passive  immunization,  738 — Technic  of  subcutaneous  inoculation,  740 — 
Technic  of  intramuscular  inoculation,  742 — Technic  of  intravenous  inoculation, 
742 — Technic  of  subdural  inoculation,  746.  Serum  Treatment  and  Prophylaxis 
of  Diphtheria,  754.  Serum  Treatment  and  Prophylaxis  of  Tetanus,  772.  Se- 
rum Treatment  and  Prophylaxis  of  Dysentery,  782.  Serum  Treatment  and 
Prophylaxis  of  Hog  Cholera,  784.  Serum  Treatment  of  Snake  Bites,  786. 
Serum  Treatment  and  Prophylaxis  of  Hay-fever,  786.  Serum  Treatment  and 
Prophylaxis  of  Meningococcus  Meningitis,  788.  Serum  Treatment  of  Influenzal 
Meningitis,  802.  Serum  Treatment  of  Pneumococcus  Meningitis,  804.  Serum 
Treatment  of  Other  Localized  Pneumococcus  Infections,  807.  Serum  Treat- 
ment of  Lobar  Pneumonia,  807.  Serum  Treatment  of  Streptococcus  Infections, 
813.  Serum  Treatment  of  Gonococcus  Infections,  818.  Serum  Treatment  of 
Staphylococcus  Infections,  819.  Serum  Treatment  of  Anthrax,  820.  Serum 
Treatment  of  Typhoid  Fever,  821.  Serum  Treatment  of  Plague,  822.  Serum 
Treatment  of  Cholera,  823.  Serum  Treatment  of  Tuberculosis,  824.  Treatment 
of  Infectious  Diseases  with  Specific  Serum  of  Convalescents  and  with  Normal 
Serum,  825.  Serum  Treatment  of  Scarlet  Fever,  825.  Serum  Treatment  of 
Acute  Anterior  Poliomyelitis,  826.  Serum  Treatment  of  Pneumonia,  828. 

CHAPTER  XXXI. — SERUM  THERAPY  (continued) 829 

Normal  Serum  Therapy,  829 — Serum  treatment  of  hemorrhage,  829— ^Serum 
treatment  of  the  toxicoses  of  pregnancy,  833 — Serum  treatment  of  skin  diseases, 
834.  Autoserum  Therapy,  835— Autoserum  treatment  of  skin  diseases,  835 — 
Autoserum  treatment  of  acute  infectious  diseases,  835 — Autoserum  treatment 
of  syphilis  (salvarsanized  serum),  836 — Autoserum  treatment  of  tuberculosis 
of  serous  membranes,  841 — Autoserum  treatment  of  non-tuberculous  effusions, 
843. 

CHAPTER  XXXII. — CHEMOTHERAPY 844 

Principles  of  Chemotherapy,  845 — Organotropism  and  parasitotropism,  845 
— Chemoreceptors,  847 — Drug  "fastness,"  849— Therapia  magna  sterilisans,  851. 
Salvarsan  and  Neosalvarsan  in  the  Treatment  of  Syphilis,  852 — Historic,  852 — 
Properties  of  salvarsan,  854 — Toxicity  of  .salvarsan,  855 — Properties  of  neo- 
salvarsan,  856— Methods  for  preparing  salvarsan  for  administration-,  857 — 
Methods  for  preparing  neosalvarsan  for  administration,  857 — Administration  of 
salvarsan  and  neosalvarsan  by  intravenous  injec^n,  858 — Reactive  manifesta- 
tions, 867 — By  intramuscular  injection,  875 — By  intraspinous  injection,  876 — 
Contraindications  and  precautions  in  salvarsan  therapy,  878 — Value  of  salvarsan 
and  neosalvarsan  in  the  treatment  of  syphilis,  879.  Salvarsan  in  the  Treatment 
of  Non-syphilitic  Diseases,  880.  Chemotherapy  in  Bacterial  Diseases,  881. 
Chemotherapy  in  Malignant  Diseases,  887. 

PART  V 
EXPERIMENTAL  INFECTION  AND  IMMUNITY 

Introductory,  890 — Methods,  890 — The  Student,  890 — Records,  891— Animal 

Experiments  and  Autopsies,  891. 

EXERCISE  1. — Active  Immunization  of  Animals,  891. 

Experiment  1,  Production  of  Agglutinins,  .Bacteriolysins  and  Opsonins,  891 — 
Experiment  2,  Production  of  Precipitins,  892— Experiment  3,  Production  of 
Hemolysins,  892 — Experiment  4,  Production  of  Cytotoxin,  892. 

EXERCISE  2. — Infection,  892. 

Experiment  5,  Experimental  Pneumonia,  892. 

EXERCISE  3. — Toxins,  893. 

Experiment  6,  Diphtheria  Toxin,  893 — Experiment  7,  Method  of  Testing  the 
Virulence  and  Toxicity  of  Diphtheria  Bacilli,  894— Experiment  8,  Tetanus  Toxin 
(in  vivo},  895 — Experiment  9,  Tetanus  Toxin  (in  vitro),  895. 


CONTENTS  9 

EXERCISE  4. — Toxins  (continued),  896. 

Experiment  10,  Botulism  Toxin,  896— Experiment  11,  Dysentery  Toxin,  896— 
Experiment  12,  Staphylotoxin  (in  vivo),  897 — Experiment  13,  Staphylotoxin 
(in  vitro),  897. 

EXERCISE  5. — Toxins  (continued) — Plant  and  Animal  Toxins,  898. 

Experiment  14,  Streptotoxin,  898 — Experiment  15,  Phytotoxins  (in  vivo  and 
in  vitro),  898 — Experiment  16,  Zootoxin,  Cobra  venom  (in  vivo  and  in  vitro),  899. 

EXERCISE  6. — Endotoxins  and  Aggressins,  899. 

Experiment  17,  Endotoxins,  899 — Experiment  18,  Natural  Aggressins,  900. 

EXERCISE  7. — Bacterial  Protein.  Ptomains.  Mechanical  Action  of  Bacteria,  900. 
Experiment  19,  Bacterial  Protein,  900 — Experiment  20,  Ptomains,  901 — Ex- 
periment 21,  Mechanical  Action  of  Bacteria,  902. 

EXERCISE  8. — Kinds  of  Immunity.     Natural  Immunity,  902. 

Experiment  22,  Phagocytosis  in  Natural  Immunity,  902 — Experiment  23,  Nat- 
ural Antibacterial  Immunity,  902 — Experiment  24,  Relative  Factors  in  Natural 
Immunity,  903 — Experiment  25,  Influence  of  Temperature  Upon  Natural 
Immunity,  903. 

EXERCISE  9. — Acquired  Immunity,  903. 

Experiment  26,  Acquired  Active  (Antibacterial)  Immunity,  903 — Experiment 
27,  Acquired  Passive  (Antitoxic)  Immunity,  904 — Experiment  28,  Acquired 
Passive  (Antitoxic)  Immunity,  904 — Experiment  29,  Acquired  Passive  (Anti- 
bacterial) Immunity,  904. 

EXERCISE  10. — Phagocytosis,  905. 

Experiment  30,  Phagocytosis  (macrophages),  905— Experiment  31,  Phagocy- 
tosis (microphages),  905 — Experiment  32,  Phagocytosis,  906. 

EXERCISE  11. — Phagocytosis.     Chemotaxis,  906. 

Experiment  33,  Positive  Chemotaxis,  906 — Experiment  34,  Negative  Chemo- 
taxis, 906. 

EXERCISE  12. — Opsonins,  907. 

Experiment  35,  Normal  Opsonins,  907 — Experiment  36,  Immune  Opsonin  (Bac- 
teriotropin),  908 — Experiment  37,  Hemopsonin,  908. 

EXERCISE  13. — Opsonins  (continued),  908 

Experiment  38,  Mechanism  of  Action  of  Opsonins,  908 — Experiment  39,  Specific- 
ity of  Opsonins,  909. 

EXERCISE  14. — Opsonic  Index,  910. 

Experiment  40,  Determining  the  Opsonic  Index,  910 — Experiment  41,  Quan- 
titative Estimation  of  Bacteriotropins,  910. 

EXERCISE  15. — Bacterial  Vaccines,  910. 

Experiment  42,  Preparation  of  Typhoid  Vaccine,  910 — Experiment  43,  Prepara- 
tion of  Staphylococcus  Vaccine,  911. 

EXERCISE  16. — Antitoxins,  911. 

Experiment  44,  Standardizing  Diphtheria  Antitoxin,  911 — Experiment  45, 
Standardizing  Tetanus  Antitoxin,  912. 

EXERCISE  17. — Antitoxins  (continued),  912. 

Experiment  46,  Specificity  of  Antitoxins,  912 — Experiment  47,  Nature  of  the 
Toxin-Antitoxin  Reaction.  Action  of  Anti-tetanolysin,  912 — Experiment  48, 
Antistaphylolysin,  913. 

EXERCISE  18. — Ferments  and  Antiferments,  914. 

Experiment  49,  Tryptic  Ferment  of  Leukocytes,  914 — Experiment  50,  Testing 
the  Antitryptic  Power  of  Bl<ood-serum,  914. 

EXERCISE  19. — Ferments  (continued),  915. 

Experiment  51,  Abderhalden  Sero-enzyme  Reaction  in  Pregnancy,  915. 

EXERCISE  20. — Agglutinins,  916. 

Experiment  52,  Gruber-Widal  Reaction  in  Typhoid  Fever  ("Wet"  Method), 
916— Experiment  53,  Gruber-Widal  Reaction  in  Typhoid  Fever  ("Dry" 
Method),  916. 

EXERCISE  21.— Agglutinins  (continued),  917. 

Experiment  54,  Macroscopic  Agglutination  Reaction,  917 — Experiment  55, 
Macroscopic  Agglutination  Reaction  (Kolle),  918 — Experiment  56,  Macro- 
scopic Agglutination  Reaction  (Killed  Cultures),  918. 


10  CONTENTS 

EXERCISE  22. — Agglutinins  (continued),  918. 

Experiment  57,  Group  Agglutination,  918 — Experiment  58,  Pro-agglutination 

(Agglutinoids),  919 — Experiment  59,  The  Absorption  or  Saturation  Agglutina- 
tion Reaction,  919. 
EXERCISE  23. — Agglutinins  (continued),  919. 

Experiment  60,  Hemagglutinins,  919 — Experiment  61,  Blood  Transfusion  Tests, 

920. 
EXERCISE  24. — Precipitins,  920. 

Experiment  62,  Titration  of  a  Precipitin  Serum,  920 — Experiment  63,  Titration 

of  a  Precipitin  Serum,  920 — Experiment  64,  Specificity  of  Precipitins,  920. 
EXERCISE  25. — Precipitins,  921. 

Experiment  65,  Forensic  Blood  Test,  921. 
EXERCISE  26. — Precipitins,  922. 

Experiment  66,  Lactoserums,  922 — Experiment  67,  Bacterial  Precipitins,  922— 

Experiment  68,  Noguchi  Globulin  Reaction,  922. 
EXERCISE  27. — Amboceptors  and  Complements.     Hemolysins,  923. 

Experiment  69,  Non-specific  Hemolysins,  923 — Experiment  70,  The  Hemolytic 

Influence  of  Acids  and  Alkalies,  924 — Experiment  71,  Serum  Hemolysis  in  vitro, 

924 — Experiment  72,  Serum  Hemolysis  in  vivo,  924. 
EXERCISE  28. — Amboceptor  and  Complements.     Hemolysins,  925. 

Experiment  73,  Titration  of  a  Hemolytic  Amboceptor,  925 — Experiment  74, 

Quantitative  Factors  in  Serum  Hemolysis,  925. 
EXERCISE  29. — Amboceptors  and  Complements.     Hemolysins,  926. 

Experiment  75,  Role  of  Amboceptor  and  Complement  in  Hemolysis,  926 — Ex- 
periment 76,  Specificity  of  Amboceptors,  926 — Experiment  77,  General  Proper- 
ties of  Amboceptors,  927. 
EXERCISE  30. — Amboceptors  and  Complements.     Hemolysins,  927. 

Experiment  78,  Mechanism  of  Amboceptor  Action,  927 — Experiment  79,  A 

Further  Study  of  the  Mechanism  of  Amboceptors,  928. 
EXERCISE  31. — Amboceptors  and  Complements.     Hemolysins,  928. 

Experiment  80,  Natural  Hemolysins.     Removal  of  Natural  Hemolysins,  928. 
EXERCISE  32. — Amboceptors  and  Complements,  929. 

Experiment  81. — Hemolytic  Complement,  929 — Experiment  82,  Inactivation 

and  Reactivation  of  Complement,  930 — Experiment  83,  General  Properties  of 

Complement,  930. 
EXERCISE  33. — Amboceptors  and  Complements,  931. 

Experiment  84,  Titration  of  Hemolytic  Complement,  931 — Experiment  85, 

Phenomenon  of  Complement  Fixation,  931. 
EXERCISE  34. — Antigens  for  the  Wassermann  Reaction,  932. 

Experiment  86,  Preparation  of  Antigens  for  the  Wassermann  Reaction,  932. 
EXERCISE  35. — Antigens,  932. 

Experiment  87,  Method  of  Titration  of  Antigens,  932. 
EXERCISE  36. — Wassermann  Reaction,  933. 

Experiment  88,  Anticomplementary  Action  of  Serums,  933. 
EXERCISE  37. — Wassermann  Reaction,  934. 

Experiment  89,  Wassermann  Reaction  (First  Method),  934. 
EXERCISE  38. — Wassermann  Reaction,  934. 

Experiment  90,  Wassermann  Reaction  (Second  Method),  934. 
EXERCISE  39. — Wassermann  Reaction,  935. 

Experiment  91,  Wassermann  Reaction  (Third  Method),  935. 
EXERCISE  40. — Wassermann  Reaction,  935. 

Experiment  92,  Wassermann  Reaction  (Fourth  Method),  935. 
EXERCISE  41. — Noguchi  Modification  of  the  Wassermann  Reaction,  935. 

Experiment  93,  Titration  of  Antihuman  Hemolysin,  935 — Experiment  94,  Tech- 

nic  of  the  Noguchi  Modification,  935. 
EXERCISE  42. — Wassermann  and  Noguchi  Reactions,  936. 

Experiment  95,  Comparison  of  Methods,  936. 
EXERCISE  43. — Gonococcus  Complement-fixation  Reaction,  936. 

Experiment  96.  Titration  of  Gonococcus  Antigen,  936. 


CONTENTS  11 

EXERCISE  44. — Gonococcus  Complement-fixation  Reaction,  936. 

Experiment  97,  Technic  of  the  Gonococcus  Reaction,  936. 
EXERCISE  45. — Gonococcus  Complement-fixation  Reaction,  937. 

Experiment  98,  Technic  of  the  Gonococcus  Reaction,  937. 
EXERCISE  46. — Complement  Fixation  in  the  Differentiation  of  Proteins,  937. 

Experiment  99,  Titration  of  Immune  Serums,  937. 
EXERCISE  47. — Complement  Fixation  in  the  Differentiation  of  Proteins,  937. 

Experiment  100,  Technic  of  the  Forensic  Blood  Test,  937. 
EXERCISE  48. — Venom  Hemolysis,  938. 

Experiment  101,  Venom  Hemolysis  in  Syphilis,  938. 
EXERCISE  49. — Bacteriolysis,  938. 

Experiment  102,  Pfeiffer  Bacteriolytic  Test,  938. 
EXERCISE  50. — Bacteriolysis,  939. 

Experiment  103,  Microscopic  Method  of  Measuring  the  Bacteriolytic  Power  of 

Blood,  939. 
EXERCISE  51. — Bacteriolysis,  939. 

Experiment  104,  Method  of  Measuring  the  Bacteriolytic  Activity  of  the  Blood 

in  vitro  (Method  of  Stern  and  Korte),  939. 
EXERCISE  52. — Cytotoxins,  940. 

Experiment  105,  Action  of  Nephrotoxin,  940. 
EXERCISE  53. — Miostagmin  Reaction,  940. 

Experiment  106,  Technic  of  the  Miostagmin  Reaction,  940. 
EXERCISE  54. — Anaphylaxis,  940. 

Experiment  107,  Anaphylaxis  in  the  Guinea-pig.     Specificity  of  Anaphylaxis, 

941. 
EXERCISE  55. — Anaphylaxis,  941. 

Experiment  108,  Nature  of  the  Anaphylatoxin,  941. 
EXERCISE  56. — Anaphylaxis,  942. 

Experiment  109,  Anaphylaxis  in  the  Dog,  942. 
EXERCISE  57. — Anaphylaxis,  942. 

Experiment  110,  Passive  Anaphylaxis,  942. 
EXERCISE  58. — Anaphylaxis,  942. 

Experiment  111,  Anti-anaphylaxis,  942. 
EXERCISE  59. — Anaphylaxis,  943. 

Experiment  112,  Local  Anaphylactic  Reactions,  943. 
EXERCISE  60. — Chemotherapy,  944. 

Experiment  113,  Salvarsan,  944. 


INDEX..  .  945 


LIST  OF  ILLUSTRATIONS 


Fia-  PAOB 

1.  An  Electric  Centrifuge 18 

2.  Method  of  Making  a  Simple  Capillary  Pipet 20 

3.  Method  of  Making  a  Looped  Pipet 21 

4.  Graduated  Pipets 22 

5.  Method  of  Making  a  Wright  Blood  Capsule 23 

6.  Method  of  Making  a  Vaccine  Ampule  of  Glass  Tubing 24 

7.  Method  of  Making  a  Large  Vaccine  Ampule  of  a  Test-tube 25 

8.  A  Satisfactory  Syringe 26 

9.  A  Suction  Pump 29 

10.  Method  of  Pricking  a  Finger 31 

11.  Method  of  Obtaining  a  Small  Amount  of  Human  Blood 32 

12.  Collecting  Blood  in  a  Wright  Capsule 33 

13.  Removing  Serum  from  a  Wright  Capsule 34 

14.  Method  of  Sealing  a  Wright  Capsule 34 

15.  Methods  for  Securing  Blood  by  Puncture  of  Vein 35 

16.  The  Keidel  Tube  for  Collecting  Blood 36 

16a.  Parts  of  the  Keidel  Tube 37 

17.  A  Wet-cup  for  Securing  Blood  from  Children  (Blackfan) 38 

18.  Technic  of  Spinal  Puncture 40 

19.  Method  of  Bleeding  a  Rabbit  from  the  Ear 42 

20.  A  Dissection  of  the  Neck  of  a  Rabbit  to  Show  Relation  of  the  Carotid  Artery  43 

21.  Method  of  Bleeding  a  Rabbit  from  the  Carotid  Artery 44 

22.  Method  of  Bleeding  a  Rabbit  from  the  Carotid  Artery  (Pasteur  Institute 

Method) 45 

23.  Method  of  Bleeding  a  Guinea-pig 46 

24.  A  Dissection  of  the  Neck  of  a  Sheep  to  Show  the  Relations  of  the  External 

Jugular  Vein 48 

25.  Method  of  Bleeding  a  Sheep  from  the  External  Jugular  Vein 49 

26.  Method  of  Bleeding  a  Horse  from  the  Jugular  Vein y . .  52 

27.  Method  of  Subcutaneous  Inoculation  of  a  Guinea-pig 55 

28.  Method  of  Intravenous  Inoculation  of  a  Rabbit 57 

29.  A  Dissection  of  the  Neck  of  a  Guinea-pig  to  Show  the  Relations  of  the  Ex- 

ternal Jugular  Vein 58 

30.  Method  of  Intravenous  Inoculation  of  a  Guinea-pig 59 

31.  Method  of  Intravenous  Inoculation  of  a  Rat 60 

32.  Method  of  Intravenous  Inoculation  of  a  Horse 61 

33.  Method  of  Intraperitoneal  Inoculation  of  a  Rabbit 63 

34.  A  Small  Berkefeld  Filter • 77 

35.  A  Filter 78 

36.  Abdominal  Wall  of  Guinea-pig  showing  Diphtheric  Edema Facing  114 

37.  Normal  Adrenal  Gland  of  a  Guinea-pig Facing  115 

13 


14  LIST   OF   ILLUSTRATIONS 

FIG.  PAGE 

38.  Adrenal  Gland  of  a  Guinea-pig  after  FatalDiphtheric  Intoxication.  .Facing  115 

39.  Ehrlich's  Side-chain  Theory.     Formation  of  Antitoxins 152 

40.  Theoretic  Structure  of  a  Molecule  of  Toxin  and  Toxoid 153 

41.  Ehrlich's  Side-chain  Theory.     Formation  of  Agglutinins  and  Precipitins...  .   155 

42.  Ehrlich's  Side-chain  Theory.     Formation  of  Cytolysins   (Bacteriolysins, 

Hemolysins,  etc.) 157 

43.  General  Scheme  of  Antigens  and  Their  Antibodies 165 

44.  Phagocytosis  (Macrophages) Facing  179 

45.  Phagocytosis  (Macrophages) Facing  179 

46.  Positive  Chemotaxis Facing  180 

47.  Negative  Chemotaxis Facing  184 

48.  Capillary  Pipet  for  Opsonic  Index  Determinations 199 

49.  Mixing  the  Contents  of  a  Capillary  Pipet 200 

50.  Method  of  Sealing  a  Capillary  Pipet 201 

51.  Method  of  Preparing  a  Blood  Film 201 

52.  Blood  Films  for  Phagocytic  Counts 202 

53.  Tubercle  Opsonic  Index Facing  203 

54.  An  Unsatisfactory  Film  for  Phagocytic  Count Facing  203 

55.  A  Satisfactory  Film  for  Phagocytic  Count Facing  203 

56.  An  Opsonic  Index  Chart .207 

57.  Preparation  of  a  Bacterial  Vaccine 212 

58.  A  Shaking  Apparatus 213 

59.  Capillary  Pipet  for  Counting  Bacterial  Vaccines 214 

60.  A  Satisfactory  Preparation  for  Counting  a  Bacterial  Vaccine Facing  215 

61.  An  Unsatisfactory  Preparation  for  Counting  a  Bacterial  Vaccine. .  .  .Facing  215 

62.  Instrument  for  the  Standardization  of  a  Platinum  Loop 216 

63.  Hopkins'  Tube  for  Standardizing  a  Bacterial  Vaccine 217 

64A.  A  Stock  Ampule  of  Vaccine  (Large) 218 

64B.  Stock  Bottle  of  Bacterial  Vaccine 218 

65.  A  Small  Vaccine  Ampule 219 

66.  Comer's  Automatic  Pipet 219 

67.  Theoretic  Formation  of  an  Antitoxin 226 

68.  Method  of  Administering  an  Intradermic  Injection  of  Diphtheria  Toxin  in 

the  Schick  Test 229 

69.  Showing  the  Small  Anemic  Area  Immediately  after  an  Intradermal  Injection  230 

70.  The  Schick  Test  for  Immunity  in  Diphtheria Facing  231 

71.  A  Flask  of  Diphtheria  Culture 236 

72.  A  Large  Toxin  Filter 237 

73.  Separation  of  Blood-serum Facing  239 

74.  A  Kitchens  Syringe 241 

75.  A  Battery  of  Kitchens'  Syringes 242 

76.  A  Dialysing  Cylinder  for  the  Abderhalden  Ferment  Test 267 

77.  The  Ninhydrin  Reaction  (Abderhalden  Ferment  Test) Facing  268 

78.  Theoretic  Formation  of  Agglutinins 280 

79.  Theoretic  Structure  of  Agglutinin  and  Agglutinoid 281 

80.  Diagrammatic  Illustration  of  the  Action  of  Agglutinin  and  Agglutinoids.. .  .  282 

81.  A  Satisfactory  Culture  for  the  Microscopic  Agglutination  Reaction 296 

82.  An  Unsatisfactory  Culture  for  the  Microscopic  Agglutination  Reaction 296 

83.  A  Positive  Agglutination  (Widal)  Reaction  in  Typhoid  Fever 300 

84.  Microscopic  Agglutination  Test  with  Dried  Blood Facing  300 

85.  Macroscopic  Agglutination  Reaction 302 

86.  Macroscopic  Agglutination  Test.     Pro-agglutination 303 


LIST    OF    ILLUSTRATIONS  15 

FIG.  PAGE 

87.  Agglutinoscope 304 

88.  The  Noguchi  Butyric  Acid  Test  for  Globulins 324 

89.  Hemin  Crystals Facing  326 

90.  Precipitin  Test.     Preparation  of  Extract  of  Blood-stains Facing  327 

91.  Uhlenhuth's  Filter 328 

92.  A  Rack  for  Precipitin  and  Agglutination  Reactions '329 

93.  Titration  of  a  Precipitin  (Serum) : 330 

94.  A  Precipitin  Reaction.     Biologic  Blood  Test 332 

95.  Theoretic  Formation  of  Cytolysins 340 

96.  Theoretic  Structure  of  a  Polyceptor 343 

97.  Scheme  Showing  Mechanism  of  Complement  Fixation 355 

98.  Theoretic  Structure  of  a  Bacteriolytic  Amboceptor 362 

99.  Method  of  Removing  Exudate  from  the  Peritoneal  Cavity  of  a  Guinea-pig  368 

100.  Culture  of  Cholera  Undergoing  Bacteriolysis.    A  Positive  Pfeiffer  Reaction  369 

101.  Culture  of  Cholera  Before  Bacteriolysis 369 

102.  Stained  Preparation  of  Cholera  Undergoing  Bacteriolysis Facing  369 

103.  Stained  Preparation  of  Cholera  Before  Bacteriolysis Facing  369 

104.  Bactericidal  Test  (Looped  Pipet  Method  of  Wright) Facing  379 

105.  Theoretic  Structure  of  a  Hemolytic  Amboceptor 386 

106.  Titration  of  Hemolytic  Amboceptor Facing  399 

107.  Venom  Hemolysis Facing  409 

108.  A  Vial  to  Contain  Blood  for  the  Wassermann  Reaction 433 

109.  Outfit  for  Collecting  Blood  for  the  Wassermann  Reaction 434 

110.  Titration  of  Hemolytic  Complement Facing  437 

111.  Titration  of  Antigen  for  Anticomplementary  Unit Facing  454 

112.  Titration  of  Antigen  for  Antigenic  Unit Facing  454 

113.  Wassermann  Reaction  (First  Method) .Facing  462 

114.  Reading  the  Wassermann  Reaction Facing  462 

115.  Wassermann  Reaction  (Second  Method) .Facing  468 

116.  Wassermann  Reaction  (Third  Method) Facing  469 

117.  Wassermann  Reaction  (Fourth  Method) Facing  475 

118.  Titration  of  Anti-human  Hemolytic  Amboceptor .Facing  479 

119.  Noguchi  Modification  of  the  Wassermann  Reaction Facing  479 

120.  Anticomplementary  Titration  of  a  Gonococcus  Antigen Facing  506 

121.  Gonococcus  Complement-fixation  Reaction Facing  508 

122.  Colloidal  Gold  Reactions. Facing  558 

123.  Urticarial  Rash  of  Serum  Sickness Facing  611 

124.  Multiform  Rash  of  Serum  Sickness Facing  611 

125.  Local  Serum  Anaphylactic  Reactions Facing  612 

126.  Method  of  Performing  the  Cutaneous  Tuberculin  Test  (von  Pirquet) 638 

127.  A  Positive  Cutaneous  Tuberculin  Reaction  (von  Pirquet) Facing  639 

128.  A  Positive  Conjunctival  Tuberculin  Reaction  (Wolff-Eisner-Calmette) 

Facing  639 

129.  A  Positive  Percutaneous  Tuberculin  Reaction  (Moro) Facing  640 

130.  A  Positive  Luetin  Reaction Facing  645 

131.  Production  of  Cow-pox  Vaccine 673 

132.  Method  of  Vaccination  Against  Smallpox 676 

133.  Vaccinia  (7-day  lesion) Facing  677 

134.  Vaccinia  (9-day  lesion) Facing  677 

135.  Vaccinoid.     A  Vaccination  Scar. Facing  677 

136.  Preparation  of  Rabies  Vaccine 686 

137.  Preparation  of  Tuberculin ' 717 


16  LIST    OF    ILLUSTRATIONS 

Fia.  PAGE 

138.  Method  of  Subcutaneous  Injection 741 

139.  Method  of  Intravenous  Injection  by  Means  of  a  Syringe 744 

140.  Method  of  Intravenous  Injection  by  Gravity 745 

141.  Outfit  for  Intraspinal   Injection  of  Antimeningitis   Serum  by   Gravity 

(Sophian) 750 

142.  Method  of  Intraspinal  Injection  by  Gravity 751 

143.  Method  of  Intraspinal  Injection  by  Means  of  a  Syringe 753 

144.  Blood  of  Rat  Infected  with  Sp.  recurrentis  (dark  ground  illumination). . . .  851 

145.  Same  After  Administration  of  Salvarsan  (dark  ground  illumination) 852 

146.  Method  of  Intravenous  Injection  of  Salvarsan 862 

147.  Method  of  Intravenous  Injection  of  Neosalvarsan 863 


INFECTION,  IMMUNITY,  AND  SPECIFIC 

THERAPY 


PART  I 

CHAPTER  I 

GENERAL  TECHNIC 

IN  this  chapter  simple  methods  are  described  for  preparing  capillary 
pipets  and  similar  apparatus  usually  made  in  the  laboratory,  and  a 
few  general  directions  are  given  concerning  the  preparation  of  glass- 
ware and  other  material  employed  in  the  various  methods  described 
in  succeeding  chapters  and  in  experimental  work. 

It  may  be  well  here  to  utter  a  word  of  caution  to  the  inexperienced 
against  observing  undue  haste  in  performing  the  manipulations  of  im- 
munologic  technic.  Careful  and  painstaking  work  is  essential  in  order  to 
secure  reliable  and  successful  results,  and  should  never  be  sacrificed  for 
speed,  the  latter  being  attained  only  by  experience. 


CENTRIFUGE 

1.  A  good  centrifuge  is  one  of  the  chief  requisites  of  a  laboratory 
equipment.     While  any  good  instrument  will  answer,  preference  should 
be  given  to  the  larger  types,  fitted  for  holding  both  15  c.c.  and  50  c.c. 
centrifuge  tubes,  propelled  by  electricity,  and  mounted  on  a  concrete 
block  in  the  laboratory  (Fig.  1). 

2.  The  machine  must  be  well  oiled. 

3.  The  counter  tubes  should  be  of  the  same  weight — it  is  our  cus- 
tom to  weigh  the  tubes  on  a  small  balance,  and  adjust  the  counter 
tubes  until  both  are  of  equal  weight. 

4.  The  centrifuge  tubes  should   rest   loosely  upon  a  rubber  disc 
or  wad  of  cotton  in  the  bottom  of  the  metal  tube  or  cup;    otherwise 
centrifuge  tubes  are  quite  likely  to  be  broken,  especially  if  the  machine 
is  run  at  high  speed. 

5.  The  machine  should  be  started  and  stopped  slowly,   and  un- 
necessary speed  and  long  running  time  should  be  avoided. 

6.  Never  centrifugalize  with  cotton  plugs  in  the  centrifuge  tubes. 

2  17 


18 


GENERAL   TECHNIC 


If  the  latter  must  be  sealed,  as  when  working  aseptically,  rubber  stoppers 
should  be  used.  However,  if  cotton  plugs  are  large  and  fit  tightly, 
they  may  be  prevented  from  becoming  displaced  by  passing  through 
them  two  cross-pins  in  such  manner  that  the  ends  will  rest  upon  the 
edge  of  the  tube.  The  plugs  are  thus  prevented  from  being  thrown  to 
the  bottom  of  the  tube. 


FIG.  1. — ELECTRIC  CENTRIFUGE. 

Mounted  on  a  concrete  block.     The  scales  are  for  the  purpose  of  weighing  and 
counterbalancing  the  tubes. 

7.  If  the  centrifuge  is  out  of  order,  however  slightly,  it  should  not 
be  used,  but  repaired  at  once,  or  else  it  may  be  ruined. 


PIPETS 

1.  Simple  Capillary  Pipets. — These  are  made  of  soft  glass  tubing 
in  the  following  way : 


PIPETS  19 

Tubing  having  a  caliber  of  6  mm.,  with  thin  walls,  that  does  not 
become  opaque,  brittle,  or  "run"  on  heating,  and  that  does  not  con- 
tain lead,  may  be  used.  The  question  of  alkalinity  is  also  of  importance 
in  connection  with  the  tubing.  Many  of  the  cheaper  grades  undergo 
disintegrative  changes,  which  are  accompanied  by  the  setting  free  of  al- 
kali, especially  when  the  glass  is  heated.  Glass  of  this  kind  should  be 
discarded,  as  it  may  introduce  an  element  of  error  into  our  experiments 
and  observations. 

2.  A  convenient  length  of  tubing — about  10  to  12  inches — is  chosen; 
this  will  make  two  pipets.     If  a  sufficient  length  of  tubing  for  both 
sides  is  not  available,  one  end  may  be  heated  and  drawn  out  with 
forceps,  or  a  handle  may  be  added  by  fusing  to  this  short  end  an  odd 
piece  of  glass. 

It  is  convenient  to  have  on  hand  a  supply  of  tubes  cut  to  correct 
lengths,  plugged  at  each  end  with  a  ball  of  cotton,  and  sterilized  in  a 
hot-air  sterilizer.  They  are  then  ready  to  be  drawn  out  as  needed, 
thus  furnishing  sterile  pipets  with  cotton  plugs  that  tend  to  prevent 
contamination. 

3.  The  flame  must  be  so  regulated  as  to  play  upon  only  so  much  of 
the  tube  as  will  suffice  to  furnish  the  glass  required  for  drawing  out  the 
tubing.     If  a  Bunsen  flame  is  used,  the  tip  of  the  inner  greenish  flame 
should  be  applied.     The  margins  of  the  flame  are  the  hottest,  and  for 
this  reason  the  tube  must  be  shifted  from  side  to  side  and  be  constantly 
rotated. 

4.  In  order  to  secure  uniform  heating  and  satisfactory  pipets  the 
tube  must   be  kept   constantly  rotated  from   the  moment   it   enters 
until  it  leaves  the  flame.     The  two  ends  of  the  tube  are  to  rest  upon  the 
middle  finger  of  each  hand  while  the  thumb  and  forefinger  hold  the 
tube  in  position  at  either  side  and  impart  the  rotatory  movement.     It 
is  also  necessary  that  the  tube  be  displaced  laterally  from  time  to  time, 
so  as  to  bring  each  portion  of  the  middle  segment  of  the  tube  in  turn  into 
the  edge  of  the  flame  (Fig.  2).     If  the  latter  precaution  is  omitted,  we 
shall  obtain  a  pipet  with  a  central  bulb  or  thicker  segment  and  with 
thinner  segments  on  each  side  corresponding  to  the  portions  of  the  tube 
which  lie  in  the  edges  or  hottest  portion  of  the  flame. 

5.  The  tube  is  heated  in  this  manner  until  the  glass  is  quite  plastic. 
No  attempt  is  made  to  draw  out  the  tube  until  it  has  been  entirely 
withdrawn  from  the  flame,  as  otherwise  a  portion  becomes  unduly  thin 
and  plastic  and  divides,  leaving  a  small,  bent,  and  very  poor  pipet  in 
each  hand. 


20  GENERAL   TECHNIC 

The  rapidity  and  force  with  which  the  tube  is  drawn  out  determine 
the  caliber  of  the  capillary  stem.  By  drawing  rapidly  a  tapering  cap- 
illary tube  is  obtained;  by  drawing  slowly  a  larger  capillary  tube  of 
more  uniform  caliber  is  obtained.  Of  course,  the  worker  cannot  take 
too  much  time,  as  the  glass  hardens  quickly.  With  a  little  practice 
this  part  of  the  technic  is  soon  mastered.  Thorough  and  uniform  heat- 
ing and  careful,  steady  pulling  when  the  tube  is  sufficiently  plastic  are 
of  primary  importance. 

When,  owing  to  an  error  in  judgment  in  heating  the  tube,  it  is  with- 
drawn before  it  is  sufficiently  plastic  and  begins  to  harden,  the  situa- 
tion cannot  be  remedied  by  drawing  out  the  tube  quickly  with  a  jerk. 
Similarly,  when  a  tube  has  been  partially  drawn  and  hardens  it  cannot, 
as  a  rule,  be  reheated  and  drawn  out  to  make  a  satisfactory  pipet. 


FIG.  2. — METHOD  OF  MAKING  A  SIMPLE  CAPILLARY  PIPET. 

Shows  manner  of  holding  tubing  in  a  flame  and  drawing  into  capillary  tubes, 
large  portion  has  been  removed  from  the  center. 


6.  After  drawing  out  the  pipets  the  hands  should  be  held  steady 
for  a  few  seconds,  i.  e.,  until  the  glass  has  hardened;    otherwise  the 
tubes  will  bend  and  be  less  satisfactory. 

7.  Capillary  pipets  are  manipulated  with  rubber  teats,  which  should 
be  of  the  best  soft  vulcanized  rubber,  and  should  fit  snugly  upon  the 
pipet,  rendering  it  air-tight. 

2.  Looped  Pipets.— Looped  pipets  find  their  main  application  in 
the  measurement  of  the  bactericidal  power  of  the  blood,  after  the 
method  of  Sir  A.  Wright.1 

The  essential  features  of  these  pipets  are:  (a)  The  capillary  stem, 
which  serves  for  measuring  and  mixing  the  bacterial  emulsion  and  serum; 
(6)  the  portion  that  serves  first  as  a  chamber  for  the  sterile  nutrient 
broth  and  later  as  a  cultivation  chamber  for  determining  whether  the 

1  "Technique  of  the  Teat  and  Capillary  Glass  Tube,"  1912.  Constable  and 
Company,  London. 


PIPETS 


21 


microbes  that  have  been  mixed  with  the  serum  have  or  have  not  been 
killed  by  it;  (c)  the  glass  loop,  which  acts  as  a  trap,  preventing  ex- 
traneous contamination;  and  (d)  the  handle,  upon  which  the  rubber 
teat  can  be  fitted.  With  a  little  practice  these  pipets  are  readily  made. 

1.  Select  glass  tubing  about  6  inches  in  length. 

2.  Heat  one  portion  about  two  inches  from  the  end,  and  when 
sufficiently  plastic,  draw  it  out  for  about  two  or  three  inches  or  until  it 
is  long  enough  to  give  a  spiral  loop  of  the  desired  dimensions  (Fig.  3). 
While  the  glass  is  still  plastic  hold  the  left  hand  steady,  and  with  the 
right  hand  lower  the  tubing  and  make  a  spiral  loop  in  such  manner 
that  the  loop  is  closely  applied,  but  does  not  touch  the  sides  of  the  upper 
and  lower  segments  of  the  tube.     Actual  contact  with  the  sides  must  be 


FIG.  3. — METHOD  OF  MAKING  A  LOOPED  PIPET. 


avoided,  for  this  would  produce  strain  and  predispose  the  tube  to 
fracture. 

3.  The  longer  portion  of  tubing  is  now  heated  and  drawn  out  to  a 
capillary  pipet  and  broken  through  at  the  desired  point. 

4.  Instead  of  this  method,  the  capillary  portion  may  be  drawn  be- 
fore the  loop  is  made. 

3.  Graduated  Pipets. — 1.  In  this  work  1  c.c.  pipets  graduated  into 
riir  c.c. ;  5  and  10  c.c.  pipets  graduated  into  yVc.c.  will  render  satisfactory 
service.  The  pipets  should  be  calibrated  close  to  the  tip,  and  should 
preferably  be  long,  with  a  narrow  lumen,  rather  than  short,  with  a  wide 
lumen,  as  the  latter  renders  the  markings  too  close  to  one  another.  For 
pipeting  small  amounts,  as  in  certain  complement-fixation  tests,  a  0.2  c.c. 
pipet  graduated  to  TTJF  c.c.  will  be  found  quite  serviceable,  permitting 


22 


GENERAL   TECHNIC 


4-\ 


7- 


4  _  GRADUATED  PIPETS. 


accurate  measurement  of  small  amounts  of 
fluid.  The  entire  length  of  these  pipets  is 
equal  to  the  ordinary  1  c.c.  pipet  which 
renders  the  subdivisions  far  apart  and  quite 
easy  to  read  (Fig.  4)  .  These  pipets  are  made 
by  competent  dealers  upon  special  request. 

2.  These    pipets    should    be    perfectly 
clean  and  clear,  sterilized,  and  have  sharp, 
easily  read  markings.     Pipets  with  broken 
tips  are  difficult  to  handle,  and  if  calibrated 
to  the  tip,  are  inaccurate. 

3.  The  worker  should  practise  methods 
of    making    accurate  measurements.     The 
slightest    slip    may    mean    an    inaccurate 
measurement  and  produce  untoward  results. 
The   mouth   end    and   the   pipeting  finger 
should   be    dry,    otherwise,    on    measuring 
small   amounts,  the  delivery  will  be  jerky 
and  usually  unsatisfactory. 

4.  After  pipets  have  held  infectious  ma- 
terial they  should  be  placed  at  once  in  a  jar 
containing   1   per   cent,   formalin   solution. 
After  pipeting    blood,  milk,  or  serum,  the 
pipets  should  be  rinsed  or  placed  in  a  jar 
containing  water  or  a  weak  lysol  solution,  as 
the  formalin  solution  tends  to  harden  these 
substances  and  renders  cleaning  quite  diffi- 
cult.    They   should   be  washed   thoroughly, 
the  mouth  end  being  plugged  neatly  and 
firmly  with  a  bit  of  cotton,  and  then  placed 
in  a  metal  box  or  wrapped  in  newspaper  and 
sterilized  in  the  hot-air  oven.     Unless  all 
the  serum,  blood,  milk,  etc.,  are  well  washed 
out,  the  pipets  may  become  occluded  and 
discolored.     If  this  occurs,  they  should  be 
soaked  for  twenty-four  hours  in  strong  nitric 
(50  per  cent.)  or  sulphuric  acid,  and  washed 
thoroughly  and  sterilized. 


These  pipets  are  gen- 
erally  used  in  immunologic 
work.  Note  the  small  cotton  plugs  in  the  mouth  ends.  The  1  c.c.  pipet  (second  from 
the  left)  is  graduated  near  to,  but  does  not  actually  include,  the  tip;  this  feature  is 
highly  desirable,  as  it  permits  measuring  small  amounts  of  fluid  and  the  pipet  is  not 
necessarily  ruined,  even  though  a  portion  of  the  tip  were  chipped  off. 


BLOOD    CAPSULES  23 

5.  The  jar  for  holding  soiled  pipets  should  contain  a  layer  of  cotton, 
otherwise  the  tip  may  be  broken  off  when  the  pipets  are  dropped  in. 

Buck1  has  recently  devised  a  multiple  pipet  capable  of  delivery 
into  twelve  test-tubes  simultaneously;  this  pipet  is  especially  service- 
able in  conducting  large  numbers  of  agglutination  and  complement- 
fixation  tests. 

BLOOD  CAPSULES 

Blood  capsules  were  devised  by  Sir  A.  Wright  for  collecting  small 
amounts  of  blood  for  examination.  The  essential  features  of  a  capsule 
are:  (a)  The  upper  straight  limb,  which  can  be  drawn  out  to  serve  as  a 
needle  for  puncturing;  (6)  the  recurved  limb,  which  makes  it  possible 
to  fill  the  capsule  by  gravity,  without  risk  of  the  inflow  being  arrested 
by  the  blood  running  down  and  blocking  the  straight  limb,  which  pro- 
vides an  outlet  for  the  air. 

These  capsules  are  easily  made  and  prove  quite  serviceable,  es- 


FIG.  5. — METHOD  OF  MAKING  A  WRIGHT  BLOOD  CAPSULE. 

pecially  for  collecting  small  amounts  of  blood  for  making  agglutination 
tests,  opsonic  measurements,  etc. 

1.  Take  a  piece  of  soft  glass  tubing  about  10  or  12  cm.  in  length,  and 
having  an  internal  diameter  of  at  least  5  mm. 

2.  Draw  one  end  out  into  a  capillary  stem,  and  break  this  at  an  ap- 
propriate point  (Fig.  5) . 

3.  Then  reinsert  the  tube  into  the  flame,  and,  leaving  a  portion  at 
least  3  cm.  in  length  to  serve  as  the  barrel  of  the  capsule,  draw  out  the 
tube  into  a  capillary  stem  about  8  cm.  in  length,  and  bend  it  so  as  to 
form  a  stout  recurved  limb  lying  in  the  horizontal  plane;   now,  before 
the  glass  has  lost  its  plasticity,  draw  the  capsule  gently  upward  so  that 

1  Jour.  Infect.  Dis.,  1916,  19,  267. 


24  GENERAL   TECHNIC 

its  long  axis  will  be  at  an  angle  of  about  30°  horizontally.  Finally,  sepa- 
rate the  capsule  from  the  main  tube  by  filing  it  across  the  capillary  por- 
tion at  the  distance  indicated  in  the  accompanying  illustration  (Fig.  5). 
4.  The  straight  limb  may  now  be  drawn  to  a  sharp  point  and  used 
as  a  needle. 

VACCINE  BULBS 

These  may  be  made  of  glass  tubing,  to  hold  1  c.c.  or  more,  al- 
though when  a  large  number  are  required,  it  is  cheaper  to  purchase 
ready-made  ampules,  such  as  are  shown  on  p.  219. 

1.  Take  a  piece  of  glass  tubing  at  least  10  cm.  in  length,  and  having 
an  internal  diameter  of  6  or  7  mm. 

2.  Seal  one  end  by  heating  it  in  a  hot  Bunsen  or  blow-pipe  flame. 

3.  After  cooling,  drop  in  one  or  two  small  glass  beads,  which  serve 
later  to  aid  in  mixing  the  vaccine  before  it  is  injected  (Fig.  6). 

4.  Heat  the  opposite  end  and  draw  out  into  a  wide  and  stout  cap- 
illary end. 

5.  This  end  may  be  broken  through;   the  whole  ampule  is  now  ster- 


FIG.  6. — METHOD  OF  MAKING  A  VACCINE  AMPULE  OF  ORDINARY  GLASS  TUBING. 

ilized  by  heat,  and  after  it  is  cooled,  the  required  amount  of  vaccine  is 
inserted. 

6.  The  open  end  is  then  sealed  in  the  flame  by  warming  the  air  in  the 
bulb  above  the  surface  of  the  liquid  and  finally  sealing  the  tip,  other- 
wise the  air  may  expand  after  heating  and  cause  an  explosion  at  the 
tip.  Care  must  be  exercised  not  to  heat  the  glass  down  to  the  level 
of  the  fluid,  as  this  tends  to  produce  steam  and  to  crack  the  bulb. 

Test-tubes  may  be  drawn  out  and  converted  into  ampules  for  hold- 
ing vaccines,  serums,  or  other  fluids. 

1.  Thin-walled   and   sterilized  test-tubes   of   appropriate   size   are 
chosen. 

2.  The  tube  is  heated  at  a  point  near  the  open  end  in  the  Bunsen 
flame,  in  the  same  manner  as  the  glass  tubing,  i.e.,  by  keeping  the  tube 
constantly  rotating  with  a  lateral  movement  to  insure  uniform  heating. 

3.  When  the  glass  has  become  plastic,  it  is  drawn  out  into  a  stout 
stem. 


TEST-TUBES 


25 


4.  After  cooling,  it  is  filed  through  at  an  appropriate  place,  being 
careful  to  leave  a  somewhat  long  stem  (Fig.  7). 

5.  The  open  end  may  now  serve  as  a  funnel  for  filling  the  bulb. 

6.  The  bulb  is  now  sealed  by  warming  the  air  above  the  level  of  the 
fluid  and  then  sealing  the  tip.     With  a  long  stem,  in  order  to  secure  a 
portion  of  the  contents,  the  sealed  end  may  be  broken  off  from  time  to 
time;  it  is  readily  resealed. 

7.  Instead  of  this  procedure  the  fluid  may  be  placed  in  the  test- 
tube  at  once,  the  upper  end  being  heated  in  the  usual  manner  and  drawn 
out;    the  stem  is  broken  through,  and  the  tip  sealed.     If  the  tube  is 
small  or  the  contents  are  such  as  will  almost  fill  a  tube,  this  method  may 
not  be  successful,  owing  to  the  production  of  steam  on  heating  the 


FIG.  7. — METHOD  OF  MAKING  A  LARGE  VACCINE  AMPULE  OF  A  TEST-TUBE. 


fluid,  which  either  cracks  the  bulb  or  causes  the  tip  to  explode  at  the 
time  of  sealing. 

TEST-TUBES 

1.  In  this  work  test-tubes  of  various  sizes  are  used  mainly  for  mak- 
ing the  agglutination,  precipitin,  complement  fixation,  and  other  tests. 
They  should  be  made  of  good  glass,  with  round  bottoms,  and  be  well 
annealed. 

2.  Test-tubes  should  be  thoroughly  clean,  clear,  and  sterilized,  pref- 
erably by  dry  heat.     It  is  not  necessary  to  plug  them  with  cotton  unless 
they  are  to  be  used  for  bacteriologic  work,  for  when  a  large  number  of 
tubes  are  used,  this  is  a  waste  of  time  and  of  material.     If  the  tubes  are 
to  be  used  within  from  twenty-four  to  forty-eight  hours,  merely  plac- 
ing the  tube  mouth  end  downward  in  the  wire  basket  is  sufficient.     A 


26 


GENERAL    TECHNIC 


piece  of  fresh  cotton  should  be  placed  in  the  oven  at  the  time  of  steriliza- 
tion, and  when  this  has  turned  a  light-brown  color,  sterilization  is  com- 
pleted. 

3.  After  a  tube  has  been  used  for  holding  living  and  infectious  or- 
ganisms, as  in  making  bacteriologic,  agglutination, 
and  opsonic  tests,  etc.,  the  tube  and  its  plug 
should  be  boiled  in  water  or  a  1  per  cent,  solution 
of  caustic  soda  before  it  is  again  handled.  In 
making  hemolytic  experiments,  the  material  is  not 
usually  infectious,  and  it  is  sufficient  to  wash  the 
tubes  without  previous  boiling.  In  every  case  all 
traces  of  acids  or  alkalis  must  be  removed  by  copious 
washing  in  plain  water,  as  the  slightest  trace  of 
1 1  either  may  interfere . with  the  accuracy  of  some 

of  the  tests.  Cheaper  grades  of  glass  may  contain 
relatively  large  amounts  of  alkali,  which  would 
introduce  a  disturbing  element  in  the  reactions. 


SYRINGES 

A  good  syringe  is  indispensable  in  perform- 
ing bacteriologic  and  immunologic  work.  Various 
sizes  should  be  at  hand,  but  usually  a  graduated 
5  c.c.  syringe  answers  most  purposes. 

1.  Many  kinds  of  syringes  are  on  the  market. 
Those  with  rubber  or  leather  plungers  and  pack- 
ings are  unsatisfactory,  as  they  cannot  be  sterilized 
by  boiling  and  soon  leak.  Nothing  is  more 
exasperating  than  a  leaking  syringe,  as  with  the 
leakage  unknown  quantities  of  inoculum  are  lost, 
not  to  mention  the  possible  dangers  of  con- 
taminating the  fingers,  the  animal,  and  the  labora- 
tory. 

Syringes  with  metal  or  glass  plungers  are  to 
be  preferred,  as  are  also   those  upon  which  the 
needle  may  be  fitted  without  screwing  (Fig.  8) . 
The  old  Koch  syringe  is  fitted  with  a  rubber  bulb  for  filling  and  ex- 
pelling the  fluid.     This  arrangement  is  well  adapted  for  making  sub- 
cutaneous injections,  but  is  somewhat  dangerous  for  purposes  of  mak- 
ing intravenous  injection,  on  account  of  the  danger  of  injecting  air. 


FIG.  8. — A  SATISFAC- 
TORY SYRINGE  (Rec- 
ord). 

This  syringe  has 
a  glass  barrel  and  met- 
al plunger.  It  is  easily 
sterilized,  durable,  and 
works  smoothly  and 
accurately. 


SOLUTIONS  27 

3.  Syringes  may  be  sterilized  by  filling  them  with  1  per  cent,  formalin 
solution  for  a  few  minutes,  followed  by  several  washings  with  sterile 
water  or  salt  solution.      This  method  is  good  for    syringes    having 
leather  or  rubber  packings  and  plungers.     It  is  not  safe  for  blood  cul- 
tures, as  spore-forming  bacteria  may  escape  the  sterilizing  process. 

4.  With  all  glass  or  metal  syringes,  it  is  best  to  boil  the  syringe, 
especially  if  a  careful  aseptic  technic  is  to  be  employed.     The  plunger 
should  be  removed  from  the  barrel,  or  else,  whether  it  be  of  glass  or 
metal,  it  will  expand  more  rapidly  than  the  accommodation  of  the  barrel 
will  permit.     All  parts  should  be  placed  in  a  pan  or  wrapped  in  gauze, 
warm  water  added,  and  boiling  allowed  to  take  place  for  a  minute  or 
so.     After  cooling  the  parts  are  adjusted. 

Wright's  method  for  sterilizing  a  hypodermic  syringe  for  the  ad- 
ministration of  vaccines  is  given  on  p.  221. 

5.  If  infectious  material  has  been  used,  the  syringe,  after  using, 
should  be  washed  out  and  sterilized.     The  needles  should  be  dried  and 
wired,  and  a  small  amount  of  vaselin  rubbed  over  to  prevent  rusting. 
The  plunger  may  likewise  be  occasionally  rubbed  with  a  small  quantity 
of  vaselin.     Needles  may  be  kept  in  oil  or  in  absolute  alcohol;   usually 
thorough  drying  and  wiring  preserves  them  in  good  condition. 


SOLUTIONS 

1.  Salt  Solution. — 0.85  per  cent,  sodium  chlorid  in  distilled  water  is 
best  adapted  for  immunologic  work.     This  solution  is  prepared  readily 
by  dissolving  8.5  grams  of  salt  in  a  liter  of  water,  filtering,  and  sterilizing 
in  an  Arnold  sterilizer  for  at  least  one  hour. 

2.  Sodium  citrate  in  1  or  2  per  cent,  solution,  made  with  normal  salt 
solution  and  not  with  plain  or  distilled  water,  is  used  to  prevent  the 
formation  of  fibrin  in  drawn  blood  and  exudates. 


CHAPTER  II 
METHODS  OF  OBTAINING  HUMAN  AND  ANIMAL  BLOOD 

As  a  general  rule,  when  blood  is  withdrawn  to  obtain  serum  a  care- 
ful aseptic  technic  should  be  employed.  Similarly,  when  erythrocytes 
are  to  be  obtained  for  purposes  of  immunization  it  is  necessary  to  avoid 
contamination  by  proper  cleansing  of  the  parts  and  the  use  of  sterile 
needles,  containers,  and  solutions.  In  obtaining  erythrocytes  for  mak- 
ing hemolytic  tests  it  is  not  necessary  that  the  blood  be  absolutely 
sterile,  the  ordinary  precautions  against  gross  contamination  being 
sufficient. 

Blood  may  be  withdrawn  to  obtain  the  corpuscles  or  serum  or  both. 

OBTAINING  CORPUSCLES 

Red  blood-corpuscles  are  usually  obtained  and  washed  free  of  serum 
for  the  purpose  of  making  hemolytic  reactions  and  experiments. 

Leukocytes  are  usually  obtained  for  the  purpose  of  estimating  the 
opsonic  index.  The  special  technic  for  obtaining  leukocytes  for  this 
test  is  given  on  p.  198. 

Larger  quantities  of  leukocytes  are  obtained  by  injecting  sterile 
irritants,  such  as  sterile  aleuronat  suspension,  into  the  pleural  or  ab- 
dominal cavities  of  suitable  animals.  (See  p:  204.) 

(a)  Blood  may  be  withdrawn  and  placed  at  once  in  two  or  three 
volumes  of  1  per  cent,  sodium  citrate  in  0.85  per  cent,  salt  solution. 
Clotting  is  prevented,  and  the  corpuscles-  are  secured  by  centrifugaliza- 
tion  or  by  sedimentation  in  the  refrigerator. 

(b)  Blood  may  be  drawn  into  a  beaker  or  flask  and  defibrinated  at 
once  by  whipping  with  a  rod  or  shaking  with  glass  beads.     When  the 
fibrin  has  been  removed,  the  corpuscles  and  serum  are  secured  by  cen- 
trifugalization. 

WASHING  ERYTHROCYTES 

For  purposes  of  immunization  or  in  making  hemolytic  tests  red 
blood-corpuscles  are  washed  free  of  serum  before  being  used. 

1.  Place  the  citrated  blood,  which  has  previously  been  diluted  with 
sufficient  salt  solution,  in  centrifuge  tubes.  Defibrinated  blood  may 

28 


WASHING    ERYTHROCYTES 


29 


be  placed  in  centrifuge  tubes  and  five  to  ten  volumes  of  sterile  normal 
salt  solution  added  and  thoroughly  mixed.  Tubes  are  then  carefully 
balanced  and  centrifuged  at  moderate  speed  for  five  minutes. 

2.  Remove  the  supernatant  fluid  down  to  the  corpuscles  with  a 
sterile  pipet.     Add  an  equal  volume  of  salt  solution;  mix  the  corpuscles 


FIG.  9. — A  SUCTION  PUMP. 

By  attaching  a  capillary  pipet  to  the  rubber  tubing  fluid  may  be  removed  without 

disturbing  the  sediment. 

and  centrifuge.  This  process  should  be  repeated  once  more  in  order  to 
insure  thorough  washing  of  the  corpuscles  to  remove  all  traces  of  serum. 
For  removing  supernatant  fluids  which  are  to  be  discarded,  the  suction 
pump  shown  in  Fig.  9  will  be  found  very  useful.  It  is  well  to  fit  the 


30         METHODS   OF   OBTAINING  HUMAN   AND   ANIMAL  BLOOD 

rubber  tubing  with  a  capillary  pipet,  which  permits  the  supernatant 
fluid  flush  with  the  sediment  to  be  removed. 

3.  After  the  last  washing  the  supernatant  salt  solution  should  be 
carefully  removed,  when  the  corpuscles  are  ready  for  use. 

OBTAINING  BLOOD-SERUM 

If  serum  is  desired  at  once,  blood  should  be  drawn  into  sterile  cen- 
trifuge tubes  and  the  tube  immersed  in  cold  water  for  from  five  to  ten 
minutes;  this  facilitates  clotting.  The  clot  is  then  broken  up  with  a 
sterile  platinum  wire  or  glass  rod,  and  the  serum  secured  by  rapid 
centrifugalization.  Or  blood  may  be  drawn  into  sterile  cylinders, 
Petri  dishes,  or  centrifuge  tubes,  and  allowed  to  stand  at  room  temper- 
ature for  a  few  hours,  after  which  they  should  be  placed  in  a  refrigerator 
until  the  serum  separates.  Blood  never  should  be  drawn  into  Erlen- 
meyer  flasks  because  of  the  difficulty  of  drawing  off  serum  without  dis- 
turbing the  clot.  When  drawn  into  Petri  dishes,  care  should  be  taken 
that  the  layer  of  blood  is  not  too  thin,  otherwise  drying  will  occur  with 
poor  separation  of  the  serum.  As  a  rule,  the  best  results  are  secured 
by  placing  blood  in  centrifuge  tubes,  for  if  separation  is  poor  or  does  not 
occur  at  all,  the  clot  may  be  broken  up  and  serum  secured  by  centrif- 
ugalization. So  far  as  possible,  avoid  drawing  blood  from  an  animal 
immediately  after  feeding,  as  under  these  circumstances  the  serum  is 
likely  to  be  milky  or  opalescent. 

OBTAINING  CORPUSCLES  AND  SERUM 

For  certain  purposes  it  may  be  desirable  to  obtain  both  serum  and 
corpuscles;  these  may  be  secured  in  the  following  way: 

1.  Place  blood  in  a  large  centrifuge  tube  or  cylinder,  and  defibrinate 
with  rods  or  glass  beads. 

2.  Centrifuge  thoroughly. 

3.  Remove  the  serum,  which  is  slightly  discolored  on  account  of 
defibrination,  with  capillary  tube  and  rubber  teat. 

4.  Filter  the  corpuscles  into  a  centrifuge  tube  through  a  wisp  of 
cotton  in  a  funnel  to  remove  small  particles  of  fibrin. 

5.  Add  normal  salt  solution,  and  proceed  with  the  washing  process. 

OBTAINING  BLOOD  PLASMA 

In  obtaining  blood  plasma  it  is  necessary  to  avoid  coagulation  of 
blood  by  securing  and  handling  the  blood  with  the  least  amount  of 


OBTAINING    BLOOD    PLASMA 


31 


trauma  to  leukocytes  (paraffined  tubes),  and  centrifuging  rapidly  and 
at  once  in  cold  centrifuge  tubes. 

1.  After  warming  a  centrifuge  tube  immerse  in  hot  molten  paraffin. 
Remove,  drain,  and  allow  the  paraffin  to  harden.     This  produces  a 
thin  coat  of  paraffin  within  the  tube;    if  a  thicker  one  is  desired,  im- 
merse again  until  a  coat  of  the  desired  thickness  is  obtained;    chill  the 
tube  thoroughly  in  cracked  ice,  but  avoid  getting  water  inside  the 
tube. 

2.  Blood  should  be  secured  quickly  by  venipuncture,  using  a  dry 
sterile  needle,  and  passing  the  blood  into  a  paraffined  tube. 


FIG.  10. — METHOD  OF  PRICKING  A  FINGER. 

The  patient's  finger  is  grasped  firmly  and  lanced  with  a  Daland  lancet  across 
the  folds  of  skin.  When  lanced  parallel  with  the  skin-folds,  the  wound  is  likely  to 
close  before  sufficient  blood  is  secured. 


3.  Centrifuge  at  once  and  at  high  speed.     If  this  is  not  possible, 
pack  the  tube  in  a  large  centrifuge  cup,  and  surround  with  finely  cracked 
ice.     This  permits  of  more  prolonged  centrifugalization,  and  at  lower 
speed,  and  yields  a  hemoglobin-free  plasma. 

4.  The  clear  yellow  plasma  is  removed  at  once  with  pipet  and  nipple. 


32         METHODS   OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 

OBTAINING  SMALL  AMOUNTS  OF  HUMAN  BLOOD 
For  obtaining  small  amounts  of  blood — up  to  2  or  3  c.c. — for  the 
Widal  reaction,  complement  fixation,  and  other  tests,  the  following 
method  is  satisfactory: 

1.  Wash  the  last  joint  of  the  middle  finger  with  alcohol.     If  the 
hand  is  cold,  it  should  be  warmed  by  immersing  it  in  hot  water.     Before 
puncturing  compress  the  finger  and  squeeze  in  such  a  manner  as  to 
drive  the  blood  toward  the  end  of  the  finger. 

2.  Prick  deeply  with  a  broad  blood  lancet,  Hagedorn  needle,  or 
scalpel  (Fig.  10). 


FIG.  11. — METHOD  OF  SECURING  A  SMALL  AMOUNT  OF  HUMAN  BLOOD. 
By  pricking  the  finger  deeply  across  the  lines  of  the  skin  with  a  broad  lancet, 
two  or  more  cubic  centimeters  of  blood  are  easily  collected  in  a  small  test-tube.     Do 
not  use  a  large  tube,  as  blood  may  be  lost  on  the  sides  of  the  tube. 


3.  Collect  the  blood  in  a  small  test-tube, — about  8  cm.  by  1  cm., 
— such  as  is  used  in  performing  the  Noguchi  reaction  for  the  serum 
diagnosis  of  syphilis  (Fig.  11). 

4.  By  squeezing  the  finger,  sufficient  blood  can  usually  be  obtained 
from  one  puncture  practically  to  fill  a  tube  of  the  size  mentioned.     One 
to  two  cubic  centimeters  of  serum  are  easily  obtained  in  this  manner,  and 
this  is  sufficient  for  cond acting  the  ordinary  serum  reactions.     When 


OBTAINING   LARGE    AMOUNTS    OF   HUMAN    BLOOD 


33 


the  treatment  of  syphilis  is  being  guided  by  the  Wassermann  reaction, 
frequent  tests  are  necessary,  and  a  patient  may  object  to  submitting  to 
repeated  venipuncture.  The  method  for  securing  blood  just  described 
is  so  simple  and  efficient  that  objections  to  it  are  never  made. 

5.  Blood  may  also  be  drawn  in  a  Wright  capsule,  made  by  drawing 
out  ordinary  thin  glass  tubing  in  the  Bunsen  burner.     (See  p.  23.) 
After  sufficient  blood  has  been  collected  (Fig.  12),  the  straight  empty 
end  is  sealed  with  a  flame  and  then  cooled  (Fig.  14) .     The  blood  is  then 
shaken  into  this  sealed  end,  and  the  bent  end,  in  turn,  sealed  with 
the  flame.     Care  should  be 

taken  not  to  .heat  the 
blood.  When  the  serum 
has  separated,  the  tube  is 
opened  by  filing  at  a  point 
above  the  clot  and  breaking, 
protecting  the  hands  with 
a  towel.  The  serum  is 
carefully  removed  with  a 
capillary  pipet  and  nipple 
(Fig.  13). 

6.  To  obtain  blood  from 
infants  and  small  children 
the  large  toe  may  be  punc- 
tured, but  as  a  rule  better 
results  are  obtained  by  wet- 
cupping  or  by  puncturing  a 


FIG.  12. — COLLECTING  BLOOD  IN  A  WRIGHT 
CAPSULE. 


vein. 


OBTAINING  LARGE  AMOUNTS  OF  HUMAN  BLOOD 
Larger  quantities  of  human  blood  may  be  required  for  making  com- 
plement fixation  reactions,  the  Abderhalden  ferment  test,  etc. 

(a)  Phlebotomy. — 1.  In  adults,  a  prominent  vein  at  the  elbow,  such 
as  the  median  basilic,  is  usually  chosen.  In  children  less  than  a  year 
old  this  vein  is  not  suitable,  better  results  being  obtained  when  the  ex- 
ternal jugular  or  a  temporal  vein  is  used. 

2.  Place  a  rubber  tourniquet  or  a  few  firm  turns  of  a  wide  muslin 
bandage  above  the  elbow. 

3.  Apply  tincture  of  iodin  to  the  skin  over  the  vein.     The  vein  may 
be  rendered  more  prominent  by  directing  the  patient  to  open  and  close 
the  hand  several  times. 

3 


34 


METHODS    OF   OBTAINING   HUMAN   AND    ANIMAL   BLOOD 


4.  Steady  the  skin  over  the  vein,  and  insert  the  needle  in  the  direc- 
tion of  the  blood-current  (Fig.  15).  It  is  more  awkward,  and  of  no 
practical  advantage,  to  puncture  in  a  downward  direction  toward  the 
hand.  The  needle  should  be  sharp,  and  of  a  size  midway  between  the 
ordinary  hypodermic  and  a  large  antitoxin  needle,  as  the  former 
is  too  small  and  the  latter  is  unnecessarily  large.  The  blood  is  then  al- 
lowed to  drop  into  a  sterile  tube.  It  is  not  necessary  to  attach 


FIG.  13. — REMOVING  SEBUM 
FROM  A  WRIGHT  CAPSULE. 


FIG.  14. — METHOD  OF  SEAL- 
ING A  WRIGHT  CAPSULE. 


a  syringe,  although  5  to  10  c.c.  of  blood  are  obtained  more  quickly 
by  this  means  on  account  of  the  possible  gentle  suction.  Needle  and 
syringe  should  be  'sterilized  by  boiling.  When  larger  quantities  of 
human  serum  are  required  as  in  auto-serum  therapy,  a  platinum- 
iridium  needle  should  be  used,  as  coagulation  in  the  needle  is  less  likely 
to  occur;  besides,  these  needles  are  readily  sterilized  by  heating  in  the 
flame. 


OBTAINING    LARGE    AMOUNTS    OF   HUMAN    BLOOD 


35 


5.  Loosen  the  tourniquet,  withdraw  the  needle  quickly,  and  seal  the 
wound  with  a  touch  of  flexible  collodion. 


FIG.  15. — METHODS  FOR  SECURING  BLOOD  BY  PUNCTURE  OF  A  VEIN. 
The  middle  figure  shows  distention  of  the  veins  of  the  arm  about  the  elbow.  The 
needle  is  entered  by  a  quick  upward  thrust.  Practically  any  prominent  and  firm 
vein  may  be  used.  The  upper  right-hand  figure  shows  collection  of  blood  in  a  test- 
tube.  Usually  10  c.c.  or  more  are  easily  collected  before  clotting  occurs.  To  secure 
large  amounts,  use  a  larger  needle  with  a  smooth  bore  (preferably  a  platinum-iridium 
needle).  The  lower  right-hand  figure  shows  collection  of  blood  in  a  Keidel  tube. 

6.  Instead  of  a  syringe,  the  5  c.c.  vacuum  bulb  devised  by  Keidel 
has  proved  quite  satisfactory  (Fig.  16).     This  apparatus  consists  of  a 


36 


METHODS    OF    OBTAINING   HUMAN   AND    ANIMAL   BLOOD 


5  c.c.  ampule  with  arm  drawn  out  to  a  capillary  tip  and  sealed  after  a 
vacuum  has  been  created  by  heating  (Fig.  16a,  B).  A  short  piece  of  rubber 
tubing  connects  the  needle  and  the  capillary  portion  of  the  ampule.  A 
needle  of  No.  25  gage  is  fitted  tightly  into  the  free 
end  of  the  rubber  tubing.  A  slender  glass  tube 
closed  at  one  end  and  flaring  slightly  at  the  other 
serves  as  a  protection  for  the  needle,  which  it  covers 
when  the  apparatus  is  sterilized.  The  apparatus  is 
sterilized  in  a  hot-air  oven  at  150°  C.  for  one  hour. 
To  obtain  a  specimen  of  blood  the  needle  is  inserted 
into  a  vein,  and  the  capillary  end  of  the  ampule 
crushed  with  a  hemostat  through  the  rubber  tubing, 
blood  flowing  into  the  ampule  and  replacing  the  vac- 
uum. The  protecting  glass  tubing  is  then  replaced. 
Not  infrequently,  especially  in  children  and  in 
obese  adults,  one  fails  to  enter  a  vein.  Several 
attempts  to  do  so  may  result  in  ruining  one  or  more 
of  the  tubes.  My  colleague,  Dr.  Alfred  Reginald 
Allen,  has  devised  a  useful  modification  in  the 
technic  of  using  this  handy  tube;  this  consisting 
in  detaching  the  bulb  from  the  rubber  tubing  and 
needle,  inserting  the  latter  into  the  vein,  and,  when 
the  blood  appears,  quickly  attaching  the  bulb  and 
breaking  the  neck  with  a  hemostat,  in  the  usual 
manner.  By  this  method  the  bulb  is  not  broken 
until  one  is  sure  he  has  entered  a  vein  and  secured  a 
specimen  of  blood. 

(&)   Wet  Cupping. — 1.    This  method   is   partic- 
ularly applicable  for  securing  blood  from  infants. 

2.  Cleanse  an  area  over  the  back  just  below  the 
angle  of  the  scapula. 

3.  Scarify  with  a  few  superficial  linear  incisions 
or  with  a  special  scarifier. 

4.  Apply  a  cup  and  exhaust  the  air  with  special 
syringe.     The  vacuum  produces  marked  congestion 
of  the  skin  with  a  ready  flow  of  blood. 

5.  Carefully  release  the  cup  and  pour  blood  into  a  tube. 

6.  The  apparatus  devised  by  Blackfan,  and  shown  in  the  accom- 
panying illustration  (Fig.  17),  is  quite  satisfactory  and  collects  blood 
in  a  sterile  tube. 


FIG.  16. — THE  KEI- 
DEL  TUBE  FOR 
COLLECTING 
BLOOD.  (Manu- 
factured by  the 
Steele  Glass  Co., 
of  Philadelphia.) 


METHOD    OF   SECURING    CEREBROSPINAL   FLUID 


37 


(c)  Placental  Blood. — For  purposes  of  immunization  corpuscles  may 
be  obtained  by  collecting  placental  blood. 

1.  After  tying  and  cutting  the  cord,  the  placental  end  is  placed  care- 
fully in  a  150  c.c.  flask  or  bottle  containing  from  25  to  50  c.c.  of  sterile 
2  per  cent,  sodium  citrate  in  physiologic  salt  solution.     To  avoid  con- 
tamination, the  cord  may  be  lightly 

sponged  with  1  per  cent,  formalin 
solution  and  severed  with  sterile 
scissors. 

2.  By  exerting  pressure  on  the 
uterus  blood  may  be  squeezed  out 
of  the  placenta.     The  flask  is  then 
sealed  with  a  sterile  cotton  plug  and 
gently  shaken. 

3.  The  corpuscles  are  obtained 
by  centrifugalization  or  sedimenta- 
tion. 


D. 


I 

METHOD  OF  SECURING  CEREBRO- 
SPINAL FLUID  (RACHICENTESIS) 

The  chief  purpose  in  making 
spinal  puncture  is  to  obtain  and 
examine  cerebrospinal  fluid  as  an 
aid  to  the  diagnosis  of  cerebro- 
spinal diseases.  It  is  mainly  of 
value  in  neurologic  and  psychiatric 
practice,  for  the  purpose  of  securing 
fluid  for  making  the  Wassermann 
reaction,  for  a  study  of  cytologic 
changes,  alterations  in  protein  con- 
tent, and  the  like.  Not  infrequently 
the  procedure  is  required  as  an  aid 
to  establishing  a  diagnosis  of  men- 
ingeal  diseases  in  children,  particu- 
larly tuberculous  meningitis,  epi- 
demic cerebrospinal  meningitis,  meningeal  irritation, 
gitis,"  etc. 

Contraindications. — Ordinarily,  when  skilfully  performed,  spinal 
puncture  is  a  harmless  procedure.  Unless  the  necessity  for  obtaining 
fluid  is  very  urgent,  the  operation  should  not  be  done  on  persons  in  poor 


FIG.  16a. — PARTS  OF  THE  KEIDELTTTBE. 
E  is  the  vacuum  bulb  which  is  at- 
tached to  the  needle  by  a  piece  of  rubber 
tubing  (D);  the  glass  tube  (E)  covers 
the  needle  and  the  whole  is  sterilized. 


serous  menin- 


38         METHODS    OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 

physical  condition.  Kaplan  has  cautioned  against  making  lumbar 
puncture  in  the  presence  of  tumors  of  the  posterior  fossa,  particularly 
of  the  cerebellum.  When  it  is  highly  desirable  to  study  the  fluid  of 
such  cases,  2  c.c.  may  be  withdrawn,  and  immediately  replaced  with 
sterile  normal  salt  solution,  or  if  no  immediate  effects  are  observed,  the 
patient  may  be  kept  in  bed  for  the  next  twenty-four  hours. 

Preparation  of  Patient. — In  bed-fast  patients  the  puncture  may  be 


FIG.  17. — A  WET  CUP  FOR  SECURING  BLOOD  FROM  CHILDREN.     (Devised  by 

Blackfan.) 

The  cup  is  held  in  this  position  over  a  scarified  area;  air  is  exhausted  by  means  of 
a  pump  attached  to  the  rubber  tubing;  blood  collects  in  the  small  test-tube.  (Made 
by  Hynson,  Westcott  &  Dunning,  Baltimore,  Md.) 

made  at  any  time;  with  ambulatory  patients,  however,  the  most  suit- 
able time  is  late  in  the  afternoon,  so  as  to  permit  the  patient  to  rest  over- 
night. 

The  ordinary  preparations  consist  in  scrubbing  the  skin  of  the  lumbar 
region  with  green  soap  and  hot  water,  using  gauze  sponges,  followed  by 
washing  with  alcohol  and  ether.  The  area  is  then  covered  with  sterile 
gauze,  and,  just  before  the  puncture  is  made,  an  application  of  10  per 
cent,  tincture  of  iodin  is  made;  or  the  preliminary  cleansing  may  be 


METHOD    OF   SECURING   CEREBROSPINAL   FLUID  39 

omitted,  two  or  three  coats  of  the  iodin .  being  sufficient.  After  the 
fluid  has  been  secured,  the  iodin  may  be  removed  with  alcohol  and 
gauze.  The  operator's  hands  should  be  cleansed  carefully  and  washed 
in  alcohol  and  bichlorid  solution  or  weak  formalin,  or  he  may  put  on 
sterilized  rubber  gloves  before  handling  the  needle  and  performing  the 
operation  itself. 

Anesthesia. — In  the  majority  of  instances  an  anesthetic  is  not 
necessary.  In  tabes  dorsalis  and  general  paralysis  (two  conditions 
most  frequently  requiring  spinal  puncture)  the  operation  is  peculiarly 
painless.  Sick  children  are  apparently  not  greatly  disturbed,  but  in 
adults  it  may  be  necessary  to  infiltrate  the  skin  about  the  site  of  punc- 
ture with  1  per  cent,  eucain  (sterile)  or  cocain  solution.  Ethyl  chlorid  is 
much  less  satisfactory,  except  for  the  mental  effect  it  has  upon  the 
patient.  Children  may  receive  a  few  drops  of  ether.  With  nervous 
patients  it  is  good  practice  to  obviate  nervous  shock  by  adopting  a  few 
simple  precautions  against  causing  unnecessary  pain. 

Technic  of  Lumbar  Puncture. — The  patient  may  either  sit  in  a 
chair  and  bend  forward,  or  lie  on  the  left  side  on  the  edge  of  a  bed  or 
table.  In  the  case  of  sick  persons,  particularly  children,  the  latter 
position  is  necessary;  it  is  also  advisable  with  nervous  patients,  as  they 
are  likely  to  bend  backward  suddenly  or  jump  up  when  the  needle  is 
inserted,  and  I  have  known  the  needle  to  be  broken  off  at  such  a  time. 
(See  p.  752.)  The  back  should  be  arched  backward,  the  patient  bend- 
ing forward,  and  the  knees  being  drawn  up  over  the  abdomen. 

The  needle  should  be  made  of  flexible,  not  rigid,  material;  for  adults, 
a  needle  10  cm.  long,  having  a  bore  of  1  to  1.5  mm.  and  furnished  with  a 
stilet,  will  be  found  satisfactory.  For  children,  a  shorter  needle  may  be 
used,  but  the  bore  should  be  about  the  same  as  that  used  for  adults. 
The  needle  should  be  sterilized  carefully  by  boiling  in  water  for  several 
minutes. 

The  operator  now  selects  a  "soft  spot"  for  puncture.  By  running 
the  finger  along  the  spines  of  the  vertebrae,  this  will  be  found  to  be  be- 
tween the  third  and  fourth  lumbar  spinous  processes,  about  on  a  level 
with  the  posterior  superior  spines  of  the  ilia.  The  needle  is  grasped 
firmly  and  inserted  with  a  sudden  thrust,  exactly  in  the  median  line, 
and  straight  forward.  The  thrust  should  be  sufficient  to  push  the 
needle  through  the  skin  and  muscles  into  the  spinous  ligaments;  it 
may  then  be  inserted  more  slowly,  a  sudden  "give  way"  indicating 
that  the  canal  has  been  entered  (Fig.  18).  This  route  is  better  than  the 
lateral  route,  as  there  is  less  danger  of  striking  vertebral  processes  or 


40 


METHODS    OF   OBTAINING   HUMAN   AND    ANIMAL   BLOOD 


other  obstructions.  The  stilet  is  now  withdrawn.  Usually  the  first 
fluid  to  appear  is  stained  with  blood  and  should  be  collected  in  a  sep- 
arate tube.  From  5  to  10  c.c.  of  fluid  are  then  collected  in  a  second 
sterile  tube,  the  needle  is  quickly  withdrawn,  and  the  puncture  wound 
sealed  with  collodion  and  cotton  or  with  adhesive  plaster. 

It  sometimes  happens  that,   on  withdrawing  the  stilet,   no  fluid 
issues  forth.     In  this  case  the  patient  is  instructed  to  take  a  deep 


FIG.  18. — TECHNIC  OF  SPINAL  PUNCTURE. 

The  patient  is  sitting  on  the  edge  of  a  chair  and  is  bent  forward;  the  crests  of  the 
ilia  are  indicated  by  black  lines,  and  are  on  a  level  with  the  spinous  process  of  the 
fourth  lumbar  vertebra;  the  "soft  spot"  is  found  just  above.  The  needle  is  shown 
in  Figs.  141  and  142.  The  first  tube  receives  the  first  few  drops  of  fluid,  which  are 
usually  blood  tinged. 

breath,  and  if  fluid  does  not  appear  now,  the  stilet  may  be  inserted 
gently  to  dislodge  any  material  that  may  be  occluding  the  needle,  or 
the  needle  may  be  withdrawn  a  trifle  if  it  has  been  inserted  too  far,  or 
may  be  advanced  a  little  if  it  has  not  entered  the  canal.  If,  however, 
the  tap  proves  a  dry  one,  or  if  only  a  few  drops  of  blood  are  obtained, 
it  is  not  advisable  to  make  another  puncture,  as  the  second  attempt  is 
likely  to  prove  as  unsuccessful  as  the  first. 


OBTAINING   SMALL   AMOUNTS   OF   ANIMAL   BLOOD  41 

After-treatment  of  the  Patient. — Occasionally  the  needle  may  strike  a 
nerve  filament,  which  occurrence  is  followed  by  more  or  less  pain  along 
the  course  of  its  distribution;  puncture  of  the  bone  is  likely  to  be  fol- 
lowed by  pain  for  several  hours.  The  majority  of  patients  are  so  little 
affected  by  lumbar  puncture  that  no  precautions  as  regards  the  after- 
treatment  are  necessary.  As  previously  stated,  it  is  advisable  for  the 
patient  to  rest  overnight.  Sudden  release  of  pressure  or  the  nervous 
shock  may  give  rise  to  severe  headache  of  one  or  of  several  days'  dura- 
tion; persons  of  hysteric  temperament  may,  in  addition,  suffer  from 
diarrhea  and  vomiting.  Rest  in  bed,  the  application  of  ice-bags,  and 
the  administration  of  sedatives  are  usually  sufficient  to  relieve  these 
after  effects. 

Disposal  of  the  Fluid. — As  a  general  rule,  the  fluid  should  be  sent 
at  once  to  a  laboratory,  as  total  cell  counts  and  bacteriologic  cultures 
are  best  made  with  fresh  fluid.  For  the  Wassermann  reaction  it  is  not 
advisable  or  necessary  to  add  a  preservative,  as  the  fluid  will  keep  for 
several  days  in  a  good  refrigerator;  if,  however,  the  fluid  is  to  be  kept 
for  longer  periods  of  time,  0.1  c.c.  of  a  1  per  cent,  solution  of  phenol 
may  be  added  to  each  cubic  centimeter  of  fluid. 


OBTAINING  SMALL  AMOUNTS  OF  ANIMAL  BLOOD 
Rabbit. — 1.  Flip  an  ear  vigorously  with  the  hand,  and  rub  with 
xylol  and  alcohol.     The  xylol  produces  marked  congestion  and  after- 
ward should  be  carefully  removed  with  alcohol  and  water,  as  it  pro- 
duces a  low-grade  inflammatory  reaction. 

2.  Puncture  a  marginal  vein  with  a  large  needle.  The  blood  will 
flow  quickly  in  drops  and  practically  any  amount  up  to  10  c.c.  or  even 
more  may  be  collected  in  a  centrifuge  or  test-tube  (Fig.  19).  For 
making  preliminary  tests  of  serum  during  immunization  2  c.c.  of  blood 
is  usually  sufficient.  Bleeding  may  be  checked  by  making  firm  pres- 
sure over  the  puncture. 

Guinea-pig. — 1.  Blood  may  readily  be  removed  directly  from  the 
heart  by  anesthetizing  the  animal  with  ether,  and  inserting  a  sterile 
needle  into  the  heart  at  the  point  of  maximum  pulsation.  A  syringe 
for  aspiration  may  be  attached,  but  better  results  are  secured  by  ad- 
justing a  suction  apparatus.  By  means  of  a  short  piece  of  rubber  tub- 
ing the  needle  may  be  connected  to  a  test-tube  so  arranged  that  a 
partial  vacuum  is  created  by  attaching  to  a  water  suction  pump.  As 
soon  as  the  heart  has  been  entered,  blood  is  seen  to  flow  into  the  tube 


42 


METHODS   OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 


and  the  constant  suction  prevents  clot  formation  in  the  needle.     In  this 
manner  5  c.c.  of  blood  may  readily  be  obtained. 

2.  Blood  may  also  be  secured  by  aspirating  the  external  jugular  vein. 
The  vein  is  exposed  by  making  a  small  incision,  as  in  giving  intravenous 
injections. 

3.  Sufficient  blood  to  make  many  complement  fixation  tests  may  be 
secured  from  a  large  pig  by  rubbing  the  ear  vigorously  with  xylol  and 
making  a  small  incision  in  the  margin.     Bleeding  is  facilitated  by  at- 
taching a  small  test-tube  with  a  side  arm  to  a  suction  pump.     When  the 


,,- 


: 


FIG.  19. — METHOD  OP  BLEEDING  A  RABBIT  FROM  THE  EAR. 

proper  tube  is  held  firmly  over  the  ear,  5  c.c.  of  blood  may  be  obtained 
by  this  method. 

Sheep. — 1.  Small  amounts  of  blood  may  be  obtained  by  punctur- 
ing one  of  the  ear  veins. 


OBTAINING  LARGE  AMOUNTS  OF  ANIMAL  BLOOD 

Rabbit. — After  immunization  of  a  rabbit  has  been  completed,  the 

animal  is  usually  bled  to  death,  the  object  being  to  secure  the  maximum 

quantity  of  serum  in  a  sterile  condition.     Various  methods  may  be 

used.     The  animal  should  be  anesthetized  by  ether  or  high  rectal 


OBTAINING    LARGE   AMOUNTS   OF   ANIMAL   BLOOD 


43 


injection  of  a  gram  of  chloral  hydrate  in  10  c.c.  of  water,  deep  sleep  being 
induced  by  the  latter  in  from  five  to  ten  minutes,  and  lasting  for  several 
hours,  during  which  time  operative  procedures  produce  no  pain. 

First  Method  (Nuttall). — The  animal  is  fastened  to  an  operating 
board,  or,  preferably,  held  by  an  assistant,  and  the  hair  over  the  neck 
and  thorax  is  moistened  with  a  1  per  cent,  lysol  solution.  By  means 


FIG.  20. — A  DISSECTION  OP  THE  NECK  OF  A  RABBIT  TO  SHOW  THE  RELATIONS  OF 

THE  CAROTID  ARTERY. 
T,  trachea;  A,  carotid  artery;  V,  internal  jugular  vein;  V.N.,  vagus  nerve. 

of  a  sterile  knife  the  skin  is  cut  longitudinally  and  the  neck  muscles  ex- 
posed for  a  considerable  distance.  The  animal  is  then  held  upright  by 
the  assistant  over  a  sterile  dish  or  a  large  sterile  funnel,  emptying  into 
a  cylinder  or  50  c.c.  centrifuge  tube.  The  operator  stretches  the  neck 
by  carrying  the  head  backward,  and  severs  the  large  vessels  on  one  or 
both  sides  of  the  neck  with  a  sharp  sterile  scalpel  or  razor,  avoiding 


44 


METHODS    OF    OBTAINING   HUMAN   AND   ANIMAL   BLOOD 


opening  the  trachea  and  esophagus.  After  bleeding,  the  dish  is  cov- 
ered or  the  tube  plugged  and  set  aside  for  the  serum  to  separate.  This 
method  is  quite  simple,  may  be  employed  by  the  inexperienced,  and 
usually  yields  a  large  amount  of  sterile  serum. 

Second  Method. — The  animal  is  fastened  to  the  operating  board  and 
the  neck  is  stretched  by  placing  a  roller  beneath  it.  The  hair  over  the 
neck  is  clipped  close,  and  the  skin  moistened  with  alcohol  and  1  per 


FIG.  21. — METHOD  OP  BLEEDING  A  RABBIT  FROM  THE  CAROTID  ARTERY, 
SECOND  METHOD. 


cent,  lysol  solution.  The  carotid  artery  of  one  side  is  exposed  by  making 
a  straight  incision  through  the  skin  over  the  trachea  and  skinning  well 
to  one  side,  exposing  the  sternohyoid  muscles  and  external  jugular  vein. 
The  carotid  artery,  internal  jugular  vein,  and  pneumogastric  nerve  are 
to  be  found  at  the  outer  border  of  the  sternohyoid  muscles  (Fig.  20). 
By  means  of  blunt  dissection  the  artery  is  exposed  and  carefully  isolated. 
Two  small  spring  clamps  or  hemostats  are  then  applied  close  together 


OBTAINING    LARGE   AMOUNTS    OF   ANIMAL   BLOOD 


45 


at  the  distal  end,  and  the  artery  divided  between  them.  The  proximal 
end  is  then  held  with  forceps  within  the  mouth  of  a  sterile  cylinder  or 
large  centrifuge  tube.  The  wall  of  the  artery  is  incised  with  fine  scissors 
proximal  to  the  forceps,  and  the  blood  is  allowed  to  flow  into  the  ves- 
sel. The  yield  of  blood  may  be  increased  somewhat  by  exerting  pres- 
sure on  the  animal's  abdomen  and  thorax. 

To  avoid  the  risk  of  contamination  in  the  foregoing  method,  the 
apparatus  shown  in  Fig.  21  may  be  used.     The  whole  apparatus  is 


FIG.  22. — METHOD  OF  BLEEDING  A  RABBIT  FROM  THE  CAROTID  ARTERY, 
THIRD  METHOD. 

sterilized  in  the  autoclav  before  using.  After  the  artery  has  been  ex- 
posed and  isolated,  a  temporary  clamp  is  applied  to  the  proximal  end. 
A  small  incision  is  made  in  the  wall  of  the  artery,  and  the  cannula  in- 
serted and  fastened  with  a  ligature.  The  clamp  is  then  removed,  and 
blood  collected  in  a  large  tube. 

Third  Method.— The  following  method,  employed  at  the  Pasteur 
Institute  at  Paris,  has  been  found  very  useful.  The  animal — a  rabbit 
or  a  guinea-pig — is  anesthetized,  and  secured  to  an  operating-table. 
The  carotid  artery  is  carefully  and  aseptically  exposed,  and  separated 


46 


METHODS    OF    OBTAINING    HUMAN    AND    ANIMAL    BLOOD 


from  the  tissues  for  a  distance  of  at  least  one  inch;    a  ligature  is  now 
tied  securely  about  the  artery,  at  the  distal  end  of  exposure;    a  second 


FIG.  23. — METHOD  OF  BLEEDING  A  GUINEA-PIG. 

ligature  is  placed  in  position,  and  looped  loosely,  ready  to  tie  about  the 
proximal  end  (Fig.  22). 


OBTAINING   LARGE   AMOUNTS   OF  ANIMAL   BLOOD  47 

The  bottom  of  a  large  sterile  test-tube  is  heated  and  drawn  out  to  a 
fine  point,  as  shown  in  the  illustration  (Fig.  22),  and  the  tip  is  broken 
off.  The  operator  now  places  his  moistened  forefinger  under  the  ar- 
tery, elevating  it  up  and  rendering  it  taut ;  the  tip  of  the  tube  is  then 
passed  through  the  wall  into  the  interior  of  the  vessel  toward  the  heart. 
The  moment  the  vessel  is  entered  the  blood-pressure  drives  the  blood 
into  the  tube,  so  that  20  c.c.  are  soon  secured.  An  assistant  now  ties 
the  ligature  below  the  site  of  puncture;  the  tube  is  withdrawn,  and  the 
tip  sealed  in  a  flame.  The  ends  of  the  ligatures  are  cut  short  and  the 
wound  is  stitched.  Healing  usually  occurs  at  once,  and  if  subsequent 
study  of  the  blood  is  required,  the  other  carotid  and  the  femorals  can 
be  used  similarly  for  securing  it. 

Fourth  Method. — The  animal  is  fastened  to  the  operating  board,  and 
the  hair  over  the  neck  and  thorax  moistened  with  alcohol  or  lysol  solu- 
tion. The  right  thorax  is  then  incised  and  held  open  by  an  assistant. 
The  right  lung  is  seized  with  sterile  forceps  and  quickly  severed  at  the 
base  with  sterile  scalpel  or  scissors.  The  heart  is  then  punctured,  and 
the  blood  is  quickly  removed  from  the  thoracic  cavity  with  a  sterile  25 
c.c.  pipet  with  a  large  opening.  Unless  the  lung  is  removed,  it  tends  to 
float  and  block  the  end  of  the  pipet.  Everything  must  be  in  readiness, 
as  otherwise  blood  will  be  lost,  flooding  the  thoracic  cavity. 

Guinea-pig. — Pig  serum  is  usually  secured  to  furnish  complement  in 
hemolytic  tests,  and  should  be  used  within  twenty-four  or  forty-eight 
hours  after  bleeding.  Precautions  to  insure  sterility  are  not,  therefore, 
usually  necessary. 

1.  The  animal  is  anesthetized  with  ether  and  the  large  vessels  of  the 
neck  on  one  side  are  exposed  by  a  longitudinal  incision.     These  are 
severed,  and  the  blood  is  collected  in  a  Petri  dish  or  in  a  centrifuge  tube 
by  means  of  a  funnel  (Fig.  23). 

2.  By  means  of  a  sharp-pointed  scissors  the  vessels  on  one  or  both 
sides  of  the  neck  may  be  incised  transversely  at  one  cut,  inserting  the 
blade  deeply  and  close  to,  but  avoiding,  the  trachea  and  esophagus. 

Rats. — 1.  Small  quantities  of  blood  may  be  obtained  by  snipping 
off  the  tip  of  the  tail  of  the  animal  and  milking  blood  into  an  appropriate 
sterilized  tube  containing  glass  beads,  or  2  per  cent,  sodium  citrate 
solution.  In  this  manner  one  or  more  cubic  centimeters  of  blood  are 
easily  obtained,  and  at  once  defibrinated  and  injected  into  the  perito- 
neal cavities  of  other  animals,  as  in  inoculating  trypanosomes,  etc. 

2.  Large  quantities  of  blood  are  obtained  by  severing  the  large  vessels 
of  the  neck,  under  anesthesia. 


48 


METHODS   OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 


Sheep. — Blood  may  easily  be  obtained  from  a  freshly  killed  animal. 
The  first  flow  of  blood  is  discarded,  and  a  portion  of  the  remainder  is 
collected  in  a  large,  sterile,  thick-walled  flask  containing  glass  beads 


FIG.  24. — A  DISSECTION  OF  THE  NECK  OF  A  SHEEP  TO  SHOW  THE  RELATIONS  OF 

THE  EXTERNAL  JUGULAR  VEIN. 

T,  trachea;  O.M.,  omo-hyoid  muscle;  E.J.V.,  external  jugular  vein^  S.C.M., 
sterno-cleido-mastoid  muscle.  This  dissection  was  made  soon  after  natural  death 
and  shows  the  position  and  size  of  the  vein  with  the  head  held  backward  as  it  is  when 
blood  is  removed  according  to  the  technic  described  in  the  text.  When  distended, 
the  vein  is  even  larger  than  shown;  it  is  quite  superficial  and  is  usually  palpable  when 
pressure  is  made  over  the  vein  at  the  base  of  the  neck. 


OBTAINING   LARGE   AMOUNTS   OF   ANIMAL   BLOOD 


49 


By  shaking  vigorously  the  blood  is  defibrinated,  if  one  desires  to  obtain 
corpuscles,  or  the  blood  may  be  collected  in  a  cylinder  and  defibrinated 
by  whipping  with  glass  rods. 

It  is  usual,  however,  in  large  laboratories,  to  keep  a  sheep  and 
remove  the  blood  as  it  may  be  required.  Small  amounts  may  be 
obtained  from  the  ear  vein,  larger  quantities  being  secured  from  an 
external  jugular  vein  in  the  following  manner: 


FIG.  25. — METHOD  OF  BLEEDING  A  SHEEP  FROM  THE  EXTERNAL  JUGULAR  VEIN. 

The  operator  is  distending  the  vein  by  pressure  over  the  base  of  the  neck  with 
the  left  hand.  When  distended,  the  vein  can  usually  be  felt  beneath  the  skin.  The 
needle  here  shown  is  reduced  to  a  little  more  than  half  the  actual  size. 

1.  One  may  do  the  bleeding  alone,  although  the  aid  of  an  assistant 
is  usually  necessary,  especially  if  the  animal  is  large  and  vicious. 

2.  The  sheep  is  thrown  on  its  back,  and  the  head  is  held  on  the  knees 
of  an  assistant  seated  on  a  low  box  or  stool. 

3.  The  operator  may  straddle  the  animal  to -hold  down  the  fore  feet, 
although  this  is  not  necessary  unless  the  animal  is  vicious. 

4 


50         METHODS    OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 

4.  The  wool  on  the  left  side  of  the  neck  is  clipped  closely  with  scis- 
sors and  alcohol  applied. 

5.  The  operator  then  grasps  the  neck  low  down  with  the  left  hand, 
and  by  means  of  the  thumb  exerts  pressure  over  the  base  of  the  neck. 
The  external  jugular  vein  will  be  found  in  a  groove  between  the  omo- 
hyoid  and  sternomastoid  muscles  (Fig.  24).     Firm  pressure  over  the 
base  of  the  neck  usually  distends  the  vein,  which  may  be  seen  or  easily 
felt.     After  locating  the  vein,  the  pressure  should  be  released  for  an 
instant,  when  the  distention  will  disappear.     In  this  way  the  operator 
may  be  more  certain  that  he  has  located  the  vein. 

6.  A  sterile  stout  needle,  at  least  two  inches  in  length  and  provided 
with  a  trocar  and  special  shank  for  firm  grasping,  is  passed  quickly  into 
the  distended  vein  in  an  upward  and  inward  direction  (Fig.  25).     It  is 
essential  that  the  needle  be  sharp,  otherwise  it  will  be  turned  aside  by 
the  wall  of  the  vein.     The  end  of  the  needle  must  not  have  too  long  a 
bevel,  or  the  point  will  pierce  the  opposite  wall  before  the  body  of  the 
needle  is  well  within  the  vein.     The  trocar  is  now  removed,  and  blood 
collected  in  a  flask  or  bottle  and  defibrinated  with  glass  beads  and  rods. 
A  short  piece  of  rubber  tubing  may  be  attached  to  the  needle.     A  suc- 
tion apparatus  is  not  needed  because  the  flow  of  blood  is  good  so  long 
as  pressure  is  preserved  over  the  vein  at  the  base  of  the  neck. 

7.  When  the  required  amount  of  blood  has  been  secured,  pressure  is 
released  and  the  needle  quickly  withdrawn.     Bleeding  ceases  at  once, 
and  the  neck  is  then  washed  with  alcohol. 

8.  By  this  method  the  same  vein  may  be  used  over  and  over  again 
for  several  .years.     I  have  never  known  infection  to  occur,  although  the 
gradual  formation  of  scar  tissue  about  the  site  of  puncture  may  inter- 
fere with  the  operation. 

Hog. — Blood  may  be  secured  from  hogs  by  clipping  off  a  small  por- 
tion of  the  tail  with  a  sharp  razor  or  scissors,  beginning  at  the  tip. 
Bleeding  is  usually  quite  free,  but  is  easily  controlled  by  a  tourniquet 
and  bandage.  The  serum  of  hogs  immunized  against  hog  cholera  is 
secured  in  this  manner. 

Monkey. — 1.  Small  quantities  of  blood — up  to  10  or  20  c.c. — may 
readily  be  obtained  from  a  small  vein  just  beneath  the  skin  which 
crosses  over  the  inner  malleolus  at  the  ankle.  When  a  tourniquet  is 
applied,  the  vein  becomes  prominent;  the  hair  is  clipped,  and  tincture 
of  iodin  applied  over  the  skin;  a  small  needle  is  passed  into  the  vein, 
and  the  blood  collected  in  a  centrifuge  tube. 

2.  Large  quantities  of  blood  may  be  obtained  from  the  femoral  or 
external  jugular  vein  under  light  ether  anesthesia. 


OBTAINING   LARGE   AMOUNTS   OF   ANIMAL   BLOOD  51 

Dog. — 1.  Small  quantities  of  blood  may  be  obtained  in  the  following 
manner:  Apply  a  tourniquet  just  above  the  knee;  clip  the  hair  over 
the  anterior  surface  of  the  leg,  and  cleanse  with  tincture  of  iodin  and 
alcohol;  make  a  small  incision  in  the  long  axis,  exactly  in  the  median 
line;  a  fairly  large  vein  appears  at  once  just  beneath  the  skin;  by  in- 
serting an  appropriately  sized  needle,  several  cubic  centimeters  of  blood 
are  quickly  and  easily  secured.  The  wound  should  be  very  small,  and 
usually  requires  no  treatment  other  than  an  application  of  collodion  and 
cotton. 

2.  Large  quantities  of  blood  are  obtained  from  the  external  jugular 
vein  with  or  without  ether  anesthesia;  the  neck  is  shaved  and  cleansed; 
the  skin  is  incised  over  the  vein,  which  is  just  beneath  the  skin,  and  blood 
removed  with  a  sterile  needle  and  syringe.  Pressure  over  the  base  of 
the  neck  renders  the  vein  more  prominent.  In  the  case  of  large  dogs,  in- 
cision is  not  necessary,  as  it  is  easy  to  enter  the  vein  directly  through  the 
skin,  as  in  bleeding  the  sheep  from  the  external  jugular  vein  or  the 
human  from  a  vein  at  the  elbow.  Blood  may  also  be  secured  from  the 
femoral  vein  under  ether  anesthesia. 

Horse. — 1.  Small  quantities  of  blood  for  making  agglutination  and 
complement  fixation  tests  may  readily  be  secured  from  a  superficial 
vein  about  the  leg.  The  hair  is  clipped  over  the  selected  area,  and  the 
skin  sterilized  with  tincture  of  iodin.  A  tourniquet  is  applied  to  render 
the  vein  prominent,  the  vessel  is  steadied  between  forefinger  and  thumb, 
and  a  needle  quickly  inserted. 

2.  Larger  quantities  of  blood  are  secured  from  the  external  jugular 
vein.  This  operation  is  easily  conducted  in  an  aseptic  manner  and 
blood  collected  in  sterile  jars.  The  neck  about  the  region  of  the  vein, 
usually  on  the  left  side,  is  clipped,  and  a  large  area  washed  with  hot 
lysol  solution.  A  sterile  sheet  may  be  thrown  about  the  shoulders. 
The  animal  is  held  or  placed  in  specially  constructed  stalls  that  pre- 
vent him  from  backing  away  or  causing  mischief.  In  large  antitoxin 
laboratories  bleeding  is  conducted  in  special  rooms,  where  a  careful 
aseptic  operating-room  technic  may  be  observed. 

The  external  jugular  vein  is  rendered  prominent  by  exerting  pres- 
sure at  the  base  of  the  neck  by  the  application  of  a  special  tourniquet 
or  by  the  thumb  and  fingers  of  the  left  hand,  the  thumb  being  placed 
just  above  the  vein.  A  small  incision  is  made  through  the  skin,  di- 
rectly above  the  vessel. 

A  large  needle  is  passed  under  the  skin  for  a  distance  of  an  inch  or 
two  and  then  thrust  into  the  vein.  Direct  puncture  into  the  vein  is 


52         METHODS   OF   OBTAINING   HUMAN   AND   ANIMAL   BLOOD 

avoided,  as  the  needle-track  under  the  skin  closes  after  the  needle  is 
withdrawn  and  serves  to  seal  the  puncture.     The  needle  is  attached  to 


FIG.  26. — BLEEDING  A  HORSE  FROM  THE  JUGULAR  VEIN. 


sterile  rubber  tubing  that  conducts  the  blood  into  special  jars  (Fig.  26), 
In  this  manner  from  six  to  twelve  liters  of  blood  are  easily  obtained. 


CHAPTER  III 

TECHNIC  OF  ANIMAL  INOCULATION 

THIS  is  a  highly  important  part  of  immunologic  work,  as  both  for  se- 
rum diagnosis  and  for  serum  therapy  the  serum  must  be  secured  from 
animals  that  have  been  artificially  immunized.  Successful  inoculation 
requires  unremitting  care  and  thoroughness,  as  the  toxic  effects  of  the 
proteins  in  general  may  kill  an  animal  before  immunization  has  been 
completed.  No  hard  and  fast  rules  can  be  laid  down — the  weight  cf 
an  animal  and  the  reaction  to  an  injection  should  decide  the  frequency 
and  the  size  of  Subsequent  injections.  It  is  better  to  proceed  slowly 
and  gradually,  than  to  give  too  large  a  dose  at  once  and  at  too  frequent 
intervals. 

GENERAL  RULES 

1.  Select  an  appropriately  sized  syringe  that  does  not  leak  upon 
being  tested  with  water.     As  has  been  stated  elsewhere,  nothing  is 
more  unsatisfactory  than  a  leaking  syringe,  for  not  only  may  the  hand 
become  soiled,  but  an  unknown  quantity  of  inoculum  is  lost. 

2.  The  inoculum  should  be  sterile.     This  is  especially  desirable 
when  giving  intravenous  and  intraperitoneal  injections.     When  living 
cultures  of  bacteria  are  to  be  injected,  the  syringe  and  the  needle  should 
be  sterilized  in  order  to  avoid  the  introduction  of  contaminating  or- 
ganisms. 

3.  Remove  the  plunger  from  the  barrel,  and  sterilize  all  the  parts  by 
boiling  for  at  least  one  minute.     As  previously  staged,   an  all-glass 
syringe  or  a  glass  barrel  and  metal  plunger  is  the  most  satisfactory. 
(See  Fig.  8.)     The   old-fashioned   syringe  with  washers  and   rubber- 
tipped  plunger  should  find  no  place  in  a  laboratory. 

4.  After  cooling,  expel  the  water  and  load  the  syringe.     This  may 
be  done  by  drawing  the  fluid  directly  into  the  syringe  and  measuring 
the  dose  by  its  markings  or  by  pipeting  the  exact  dose  into  a  sterile 
Petri  dish  or  capsule  and  drawing  up  in  the  syringe. 

5.  The  animal  should  be  fastened  or  held  firmly  and  in  an  easy  posi- 
tion.    Everything  should  be  in  readiness,  so  that  the  injections  may  be 
given  thoroughly  and  carefully. 

53 


54  TECHNIC    OF   ANIMAL   INOCULATION 

6.  In  preparing  the  inoculum  care  should  be  exercised  that  no  solid 
particles  enter  the  syringe.     Aside  from  possibly  blocking  the  needle  and 
interfering  with  the  injection,  the  subcutaneous  injection  of  small  frag- 
ments may  do  no  particular  harm,  but  in  intravenous  inoculation  they 
may  cause  fatal   embolism.     To   obviate  this   danger  the  inoculum 
should,  if  possible,  be  filtered  through  sterile  filter-paper  before  the 
syringe  is  filled. 

7.  Air-bubbles  should  be  removed.     The  injection  of  small  bubbles 
of  air  into  subcutaneous  tissues  may  cause  no  harm,  but  when  injected 
into  veins  they  may  cause  serious  disturbances  or  immediate  death. 
To  avoid  this  the  syringe,  after  being  filled,  should  be  held  vertically, 
with  the  needle  uppermost.     The  needle  should  be  wrapped  in  cotton 
soaked  in  alcohol,  and  the  piston  of  the  syringe  pressed  upward  until 
all  the  air  is  expelled  from  the  barrel  and  the  needle.     If  a  drop  of  in- 
oculum is  forced  out,  it  will  be  collected  on  the  cotton,  which  should 
immediately  be  burned. 

8.  Injections  should  be  given  slowly. 

9.  The  animal  is  then  tagged  or  marked,  or  its  coloring  recorded.     In 
the  case  of  rabbits,  the  metal  ear  tag  is  best.     All  data,  e.  g.,  the  date, 
size  of  dose,  preparation  and  kind  of  inoculum,  etc.,  should  be  recorded 
in  writing. 

10.  When  it  is  necessary  to  incise  the  skin  in  order  to  reach  a  vein 
an  anesthetic  may  be  given.     With  superficial  veins,  and  in  subcutaneous 
inoculations,  the  injections  may  be  given  so  readily  and  easily  that  no 
more  pain  can  be  felt  than  that  which  accompanies  similar  injections  in 
human  beings. 

Animals  may  be  actively  immunized  in  a  variety  of  ways  and  in 
different  locations  in  the  anifnal  body.  For  a  particular  antibody,  a 
certain  method  may  be  found  especially  efficacious,  and  this  is  dealt 
with  in  a  subsequent  chapter.  In  serologic  work  immunization  may 
be  performed  by  subcutaneous,  intramuscular,  intravenous,  intracardial, 
and  intraperitoneal  injections. 


METHOD  OF  PERFORMING  SUBCUTANEOUS  INOCULATION 

Fluid  Inoculum. — 1.  Injections  are  usually  given  in  the  median  line 
of  the  abdominal  wall. 

2.  Have  the  animal  (a  rabbit  or  a  guinea-pig)  held  firmly  by  an 
assistant  or  secured  to  the  operating-table. 

3.  Clip  the  hair  where  injection  is  to  be  made — it  is  not  always 


METHOD  OF  PERFORMING  SUBCUTANEOUS  INOCULATION   55 

necessary  to  shave  the  area.     Apply  a  2  per  cent,  iodin  in  alcohol  solu- 
tion. 

4.  Pinch  up  a  fold  of  skin  between  the  forefinger  and  the  thumb  of 
the  left  hand;  hold  the  syringe  in  the  right  hand,  and  insert  the  needle 
into  the  ridge  of  skin  between  the  finger  and  thumb,  and  push  it  steadily 
onward  until  the  needle  has  been  inserted  about  an  inch  (Fig.  27). 
Care  must  be  exercised  not  to  enter  the  peritoneal  cavity.  Relax  the 


FIG.  27. — SUBCUTANEOUS  INOCULATION  OF  A  GUINEA-PIG. 
A  fold  of  skin  is  pinched  up  and  the  needle  entered  into  the  fold.     The  skin  is 
then  released,  and  the  injection  slowly  given.     A  swelling  indicates  that  the  injection 
is  subcutaneous. 

grasp  of  the  left  hand  and  slowly  inject  the  fluid.  If  the  skin  is  raised, 
this  shows  that  the  injection  is  subcutaneous.  If  it  is  not,  the  needle 
should  be  slightly  withdrawn  and  inserted. 

5.  Withdraw  the  needle,  and  at  the  same  time  cover  the  puncture 
with  a  wad  of  cotton  wet  with  alcohol.  A  touch  of  flexible  collodion 
over  the  puncture  completes  the  operation. 

Solid  Inoculum. — Steps  1  to  3  are  the  same  as  in  the  preceding. 


56  TECHNIC    OF   ANIMAL   INOCULATION 

4.  Raise  a  small  fold  of  skin  with  a  pair  of  forceps,  and  make  a 
tiny  incision  through  the  skin  with  a  pair  of  sharp-pointed  scissors. 

5.  With  a  probe,  separate  the  skin  from  the  underlying  muscles  to 
form  a  funnel-shaped  pocket. 

6.  By  means  of  a  fine-pointed  forceps  or  a  glass  tube  syringe  in- 
troduce the  inoculum  into  this  pocket  and  deposit  it  as  far  as  possible 
from  the  point  of  entrance  of  the  instrument. 

7.  Close  the  wound  with  collodion  and  cotton.     A  single  stitch  with 
fine  thread  may  be  necessary. 

METHOD  OF  MAKING  INTRAMUSCULAR  INOCULATION 

1.  These  injections  are  usually  made  into  the  posterior  muscles  of 
the  thigh  or  into  the  lateral  thoracic  or  abdominal  muscles. 

2.  Clip  away  the  hair  over  the  selected  area,  cleanse,  etc.,  as  for 
subcutaneous  injection. 

3.  Steady  the  skin  over  the  selected  muscles  with  the  slightly  sep- 
arated left  forefinger  and  thumb. 

4.  Thrust  the  needle  of  the  syringe  quickly  into  the  muscular  tissue-, 
and  slowly  inject  the  fluid. 

METHOD  OF  MAKING  INTRAVENOUS  INOCULATION 
Rabbit. — 1.  The  posterior  auricular  vein  along  the  outer  margin 
of  the  ear  is  better  adapted  than  a  median  vein  for  this  purpose. 

2.  If  a  number  of  injections  are  to  be  made,  commence  as  near  the 
tip  of  the  ear  as  possible,  as  the  vein  may  become  occluded  with  thrombi, 
and  subsequent  inoculations  may  then  be  given  nearer  and  nearer  the 
root  of  the  ear. 

3.  The  animal  should  be  held  firmly,  as  the  slightest  movement  may 
result  in  piercing  the  vein  through  and  through  and  require  reinsertion 
of  the  needle.     This  is  accomplished  satisfactorily  by  placing  the  rabbit 
upon  the  edge  of  the  table  and  holding  it  firmly  there  by  grasping  the 
neck  and  front  quarters,  the  assistant  at  the  same  time  compressing 
the  root  of  the  ear  with  the  thumb  and  forefinger. 

4.  If  the  hair  is  long,  clip  it. 

5.  The  ear  is  struck  gently  with  the  fingers  and  washed  with  alcohol 
and  xylol ;   the  friction  will  render  the  vein  more  conspicuous. 

6.  The  ear  is  grasped  at  its  tip  and  stretched  toward  the  operator, 
or  the  vein  may  be  steadied  by  rolling  the  ear  gently  over  the  left  in- 
dex-finger and  holding  it  between  the  finger  and  thumb. 


METHOD    OF   MAKING   INTRAVENOUS   INOCULATION  57 

7.  The  inoculum  should  be  free  from  solid  particles,  and  all  the  air 
excluded  from  the  syringe.     As  a  general  rule,  the  amount  injected 
should  be  as  small  as  possible,  and  the.  temperature  of  the  inoculum  be 
near  that  of  the  body.    If  the  syringe  is  filled  shortly  after  sterilization, 
when  it  has  cooled  enough  to  be  comfortably  hot  to  the  touch  the  heat 
will  warm  the  injection  fluid  and  not  be  hot  enough  to  cause  coagulation. 

8.  Hold  the  syringe  as  one  would  hold  a  pen,  and  thrust  the  point 
of  the  needle  through  the  skin  and  the  wall  of  the  vein  until  it  enters  the 
lumen  of  the  vein  (Fig.  28). 

9.  Direct  the  assistant  to  release  the  pressure  at  the  root  of  the  ear, 
and  slowly  inject  the  inoculum.     If  the  fluid  is  being  forced  into  the 


FIG.  28. — INTRAVENOUS  INOCULATION  OF  A  RABBIT. 

subcutaneous  tissue,  which  will  be  evident  at  once  by  the  swelling  which 
occurs,  the  injection  must  cease  and  another  attempt  be  made. 

10.  The  needle  is  quickly  withdrawn,  a  small  piece  of  cotton  moist- 
ened with  alcohol  placed  upon  the  puncture  wound,  and  firm  compression 
applied.  Wash  the  ear  thoroughly  with  alcohol  and  water  to  remove  xylol, 
otherwise  a  low-grade  inflammation  which  will  render  subsequent  injec- 
tions more  difficult  will  follow. 

Guinea-pig. — 1.  Since  the  superficial  veins  are  quite  small,  it  is 
necessary  to  make  the  injection  into  the  external  jugular  vein. 

2.  The  animal  is  tied  to  the  operating-table  and  the  hair  clipped 
away  about  the  neck  and  shoulder  on  the  right  side,  and  2  per  cent, 
iodin  in  alcohol  applied. 


58  TECHNIC    OF   ANIMAL   INOCULATION 

3.  A  small  roll  is  placed  under  the  neck  of  the  animal,  to  render  the 
operative  area  tenser  and  more  easily  accessible. 

4.  A  few  drops  of  ether  may  be  given  by  an  assistant,  although  one 
soon  learns  to  expose  the  vein  quickly  and  there  is  practically  no  pain 


FIG.  29. — A  DISSECTION  OF  THE  NECK  OF  A  GUINEA-PIG  TO  SHOW  THE  RELATIONS 

OF  THE  EXTERNAL  JUGULAR  VEIN. 

The  skin  has  been  turned  aside  and  the  superficial  fascia  and  fat  removed;  the 
position  of  the  vein  is  well  shown  and  is  readily  exposed  by  a  small  and  superficial 
incision. 

after  the  skin  has  been  incised.     If  anesthesia  is  employed,  it  should  be 
just  sufficient  to  overcome  the  struggles  of  the  animal. 

5.  The  assistant  is  directed  to  hold  the  head  backward  in  the  med- 
ian line. 


METHOD    OF   MAKING   INTRAVENOUS   INOCULATION 


59 


6.  Pick  up  the  skin  just  above  and  in  the  middle  of  the  space  be- 
tween the  shoulder  and  the  tip  of  the  upper  end  of  the  sternum — just 
above  and  about  in  the  center  of  the  area  where  a  clavicle  in  the  human 
would  be  situated.  With  sharp  small  scissors  incise  the  skin  for  about 
one-third  of  an  inch.  Separate  the  subcutaneous  tissue  gently  with 
forceps;  a  large  vein  at  once  comes  in  view  (Fig.  29).  Gently  dissect 
it  free  for  about  one-fourth  of  an  inch. 


FIG.  30. — INTRAVENOUS  INOCULATION  OF  A  GUINEA-PIG. 

The  vein  is  steadied  by  a  pair  of  fine  forceps  and  the  injection  given  through  a 
small  needle.  The  incision  here  shown  is  larger  than  actually  required  in  practice; 
the  vein  is  also  smaller  than  normal,  as  the  animal  was  dead  for  a  few  hours  prior  to 
making  the  illustration. 

7.  Pick  up  the  vein  with  a  pair  of  fine  forceps,  insert  the  needle 
of  the  syringe  gently  in  the  long  axis  of  the  vein  and  slowly  inject  the 
fluid  (Fig.  30). 

8.  Withdraw  the  needle  and  apply  firm  pressure  with  a  wad  of  clean 
gauze  or  cotton.     It  is  not  necessary  to  tie  off  the  vein.     A  stitch  may 
be  inserted  to  close  the  skin  wound  and  flexible  collodion  applied. 


60 


TECHNIC    OF   ANIMAL   INOCULATION 


Mice  and  Rats. — 1.  Mice  and  rats  may  be  injected  through  a 
caudal  vein  of  the  tail.  These  veins  are  quite  small,  and  the  injection 
requires  a  fine  needle  and  some  experience  in  the  manipulations. 

2.  Fasten  the  mouse 
*-                  -  in   a   special   trap,    so 

that  the  tail  alone  will 
be  exposed.  Grasp  the 
tip  between  the  left 
thumb  and  index- 
finger,  and  hold  the  tail 
fully  extended. 

3.  A  caudal  vein  is 
rendered  prominent  by 
the  gentle  application  of 
heat  in  the  form  of  hot 
water,  or  by  vigorous 
rubbing  with  xylol  or  al- 
cohol.    The  superficial 
cells  become  softened, 
and  may  be  scraped  off 
with   a   sharp   scalpel, 
exposing  a  vein  on  each 
side  of  the  middle  line 
of  the  tail. 

4.  It  is  usually  ad- 
visable to  have  an  as- 
sistant steady  the  tail 
while  the  inoculation  is 
being    given;     a    fine 
needle  is  essential.     In- 
oculation should  begin 
as  near  the  tip  of  the 


FIG.  31. — METHOD  OF  INTRAVENOUS  INOCULATION  OF 
a  RAT. 


The  hairs  and  superficial  layers  of  the  skin  have  tail  as  possible,  and  in 
been  scraped  away  with  a  scalpel.  The  vein  on  each  ...  ,  ,. 

side  of  the  middle  line  appears  as  a  bluish  line  in  the  subsequent  inoculations 

subcutaneous  tissues.  gradually  approach  the 

root  (Fig.  31). 

5.  Rats  may  also  be  injected  through  the  external  jugular  vein,  in 
exactly  the  same  manner  as  a  guinea-pig  is  inoculated.  (See  Fig.  30.) 
The  animal  is  fastened  to  a  small  operating  board,  and  an  assistant 
holds  the  head  to  the  left,  which  stretches  the  tissues  of  the  right  shoulder 


METHOD    OF    MAKING    INTRAVENOUS    INOCULATION 


61 


and  side  of  the  neck.  A  small  incision  is  made  midway  between  the 
middle  line  of  the  neck  and  the  tip  of  the  fore-shoulder.  With  super- 
ficial dissection  a  prominent  vein  appears;  this  vein  is  picked  up  with 
fine  forceps  and  the  injection  is  readily  given  through  a  fine  needle. 

Horse. — 1.  Horses  are  usually  injected  in  the  external  jugular  vejn. 

2.  The  hair  of  the  neck  in  the  region  of  the  site  of  inoculation  should 
be  clipped  and  thoroughly  scrubbed  with  a  hot  solution  of  lysol. 


FIG.  32. — INTRAVENOUS  INOCULATION  OF  HORSE. 

The  operator  causes  the  vein  to  distend  and  become  prominent  by  pressure  with 
the  left  hand.  The  needle  is  entered  beneath  the  skin  and  is  pushed  upward  for  an 
inch  or  more  before  the  vein  is  entered.  When  withdrawn,  this  tunneled  passage 
closes  and  prevents  bleeding.  Larger  injections  may  be  given  in  the  same  manner 
by  gravity. 

3.  The  horse  should  be  held  by  an  assistant;  if  the  animal  is  vicious, 
the  injections  should  be  given  in  a  specially  constructed  stall. 

4.  The  vein  is  distended  by  the  operator,  who  grasps  the  region  with 
his  left  hand,  the  thumb  being  directly  over  the  vein  (Fig.  32). 

5.  The  needle  is  inserted  beneath  the  skin,  and  passed  upward  for 
a  short  distance,  and  then  thrust  into  the  vein. 


62  TECHNIC    OF   ANIMAL   INOCULATION 

6.  After  the  injection  has  been  given,  either  with  a  syringe  or,  when 
the  inoculum  is  large  in  amount  by  gravity  from  a  large  jar,  the  needle 
is  quickly  withdrawn.  Bleeding  ceases  as  soon  as  pressure  over  the 
vein  is  removed. 

.  Sheep  and  Goats. — In  sheep  and  goats  the  intravenous  injection  is 
given  into  the  external  jugular  vein,  directly  through  the  skin.  The 
hair  is  clipped,  and  the  part  shaved  and  disinfected.  Compression  by 
the  finger  at  the  root  of  the  neck  renders  the  vein  more  prominent.  In- 
jections are  also  readily  given  through  a  popliteal  or  a  femoral  vein. 
If  necessary,  a  small  incision  may  be  made  through  the  skin  in  order  to 
expose  the  vein  chosen  for  the  injection. 

Dog. — Dogs  may  be  injected  through  the  external  jugular  or  pop- 
liteal veins.  The  animal  should  be  fastened  to  the  operating-table. 

2.  There  is  a  small  vein  just  beneath  the  skin,  in  the  median  line, 
along  the  anterior  surface  of  the  leg,  which  is  readily  accessible.  Clip 
away  the  hair,  and  disinfect  with  iodin  and  alcohol.  Direct  the  as- 
sistant to  grasp  the  thigh  just  above  the  knee,  to  distend  the  vein  and 
prevent  movement,  and  make  a  small  incision  directly  in  the  median 
line.  A  small  vein  is  seen  at  once.  Dissect  free  or  pick  up  gently  with 
fine  forceps  and  insert  a  small  sharp  needle.  The  injection  can  thus 
be  readily  given.  Withdraw  the  needle,  apply  firm  pressure,  and  insert 
a  single  stitch.  Bind  the  wound  with  a  few  turns  of  a  gauze  bandage 
or  seal  with  collodion  and  cotton. 

METHOD  OF  MAKING  INTRACARDIAL  INOCULATION 

1.  Guinea-pigs  may  be  injected  by  the  intracardial  route  instead  of 
intravenously.     The  technic  is  not,  as  a  rule,  more  difficult,  and  no  ill 
effects  are  noticed.     Not  infrequently,  however,  attempts  to  inject  in 
the  heart  fail,  and  frequent  trials  are  not  permissible  on  account  of 
the  danger  of  injuring  the  organ. 

2.  The  animal  is  tied  to  the  operating  board,  or  held  firmly  by  an 
assistant;   an  anesthetic  may  be  given. 

3.  Determine  the  point  of  maximum  pulsation  to  the  left  of  the 
sternum  by  palpation,  and  quickly  insert  a  thin,  sharp  needle  at  the 
selected  area.     A  flow  of  blood  indicates  that  the  needle  has  entered  the 
heart.    Attach  the  previously  filled  syringe  and  slowly  in j  ect  the  contents . 

4.  Detach  the  syringe  in  order  to  make  sure  that  the  injection  was 
intracardial,  as  intended,  which  is  indicated  by  a  flow  of  blood;   then 
quickly  withdraw  the  needle.     The  puncture  wound  may  be  sealed  with 
collodion. 


METHOD   OF   MAKING   INTRACARDIAL   INOCULATION  63 


FIG.  33. — METHOD  OF  PERFORMING  INTRAPERITONEAL  INOCULATION  OF  A  RABBIT. 
The  head  is  held  downward;  the  intestines  gravitate  toward  the  diaphragm 
(note  distention);   this  leaves  an  area  between  the  umbilicus  and  pelvis  relatively 
free  of  intestines  and  lessens  the  danger  of  puncturing  the  intestines. 


64  TECHNIC    OF   ANIMAL   INOCULATION 


METHOD  OF  MAKING  INTRAPERITONEAL  INOCULATION 
Rabbit. — 1.  Clip  the  hair  and  shave  an  area  about  two  inches  in 
diameter  in  the  median  abdominal  line,  just  below  the  umbilicus. 
Apply  2  per  cent,  iodin  in  alcohol. 

2.  Direct  an  assistant  to  hold  the  animal  firmly,  head  down.     With 
the  animal  in  this  position  the  loops  of  intestine  tend  to  sink  toward  the 
diaphragm,  leaving  an  area  above  the  bladder  which  is  sometimes  free 
of  intestines  (Fig.  33). 

3.  The  syringe  is  grasped  firmly,  and  the  needle  inserted  beneath  the 
skin  for  a  short  distance,  in  the  direction  of  the  head  in  the  long  axis  of 
the  animal  when  the  hand  is  raised  and  the  needle  forced  forward 
through  the  peritoneum.     When  the  peritoneum  has  been  entered,  this 
is  evidenced  by  a  relaxation  of  the  abdominal  muscles.     The  needle 
is  then  withdrawn  slightly  and  the  injection  made. 

Guinea-pig. — 1.  Direct  an  assistant  to  hold  the  animal  firmly  upon 
its  back.  This  is  better  than  fastening  it  to  an  operating-table,  for  it 
permits  relaxation  of  the  abdominal  wall  when  the  injection  is  to  be 
made. 

2.  Clip  the  hair  close  to  the  skin  in  the  median  abdominal  line.     A 
small  area  may  be  shaved  although  this  is  not  necessary.     Disinfect 
with  an  application  of  iodin  in  alcohol. 

3.  With  the  left  forefinger  and  thumb  pinch  up  the  entire  thickness 
of  the  abdominal  parietes  in  a  triangular  fold,  and  slip  the  peritoneal  sur- 
faces over  each  other  to  ascertain  that  no  coils  of  intestine  are  included. 

4.  Grasp  the  syringe  in  the  right  hand,  and  insert  the  needle  into 
the  fold  near  its  base. 

5.  Release  the  fold  and  inject  the  fluid.     If  a  swelling  forms,  this 
shows  that  the  needle  is  in  the  subcutaneous  tissues,  and  another  at- 
tempt should  be  made  to  enter  the  peritoneum. 

6.  It  may  be  difficult  to  pinch  up  the  parietes  without  including  the 
intestine.     In  such  case  straighten  out  the  animal  and  stretch  the  skin 
between  the  left  forefinger  and  thumb.     Insert  the  needle  obliquely 
until  it  is  beneath  the  skin.     A  slight  thrust  suffices  to  pierce  the  peri- 
toneum, when  the  abdominal  muscles  will  be  felt  to  relax.     Withdraw  the 
needle  slightly  and  inject  the  fluid. 

7.  Seal  the  wound  with  a  touch  of  collodion. 


CHAPTER  IV 

METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF 

ANIMALS 

IN  overcoming  an  infection,  acquired  either  naturally  or  artificially, 
the  macroorganism  develops  antibodies,  or  protective  substances, 
against  the  infecting  agent.  Possessed  of  these  antibodies,  the  animal 
can  subsequently  withstand  a  more  severe  attack  of  the  same  infection. 
Thus,  the  body-cells  of  the  animal  itself  are  actively  concerned  in  pro- 
ducing these  antibodies,  and  the  resulting  protection  or  immunity  is 
therefore  called  "active  immunity." 

The  general  term  "antigen"  has  been  applied  to ^nyjjubstance, that 
can  stimulate  the  formation  of  an  antibody.  The  immunity  following 
scarlet  fever  is  an  example  of  active  acquired  immunity,  although  the 
antigen  is  unknown.  In  order  to  acquire  immunity  it  is  not  always 
necessary  for  a  person  to  have  had  the  disease.  Thus,  a  severe  infec- 
tion with  the  antigen  of  typhoid  fever — Bacillus  typhosus — results  in  a 
general  reaction,  exhibiting  symptoms  and  course  known  clinically  as 
typhoid  or  enteric  fever;  whereas,  if  the  antigen  is  attenuated  and  in- 
jected artificially  in  small  doses  in  the  form  of  a  vaccine,  the  body-cells 
react,  producing  antibodies  and  a  resulting  immunity  against  typhoid 
fever,  without  discomfort  or  danger  to  life.  This  process  is  known  as 
vaccination,  the  term  being  first  applied  to  a  similar  procedure  employed 
in  inducing  an  immunity  against  smallpox  by  the  inoculation  of  a  small 
dose  of  the  antigen  attenuated  or  modified  by  passage  through  the  cow 
(cow-pox  virus) . 

This  process  of  stimulating  the  body-cells  to  produce  antibodies  is 
called  active  immunization,  and,  while  the  immunity  following  disease 
is  an  example,  the  term  is  generally  applied  to  artificial  immunization, 
as  in  vaccination,  which  serves  in  medicine,  therefore,  the  primary  pur- 
pose of  effecting  prophylaxis.  In  laboratories  active  immunization  of 
animals  is  generally  undertaken  with  a  view  to  obtaining  serums  to 
be  used  for  diagnostic  or  therapeutic  purposes. 

Practically,  any  protein  may  serve  as  an  antigen.  Thus,  animals 
may  be  immunized  not  only  with  various  bacteria,  but  with  serum  al- 
bumins and  globulins,  milk,  egg-albumen,  epithelial  cells,  etc.  It  must 
5  65 


66  METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF  ANIMALS 

be  remembered  that  these  substances — the  antigens — are  toxic,  and 
that  the  process  of  immunization  may  evoke  a  marked  disturbance  in 
the  general  health  of  the  animal.  Special  attention  should  be  given  to 
feeding  and  the  general  care  of  the  animal,  the  temperature,  weight,  the 
presence  of  diarrhea,  and  the  occurrence  of  abscesses,  edema,  paralysis, 
etc. 

If  the  animal  dies,  a  careful  postmortem  and  bacteriologic  examina- 
tion should  be  made,  in  order  to  study  the  changes  produced  by  the 
inoculated  antigen,  and  to  ascertain  if  death  was  induced  by  the  antigen 
or  by  contamination  and  secondary  infection. 

In  the  manufacture  of  serums  on  a  large  scale,  especially  for  thera- 
peutic use,  horses  are  used  almost  exclusively.  For  diagnostic  purposes 
and  especially  in  the  study  of  immunity,  smaller  animals,  such  as  rab- 
bits, guinea-pigs,  white  mice,  and  rats,  and  occasionally  goats  or  sheep, 
are  employed. 

So  far  as  their  power  of  producing  antibodies  is  concerned,  there  are 
individual  differences  among  the  same  species  of  animals;  thus,  horses 
immunized  against  diphtheria  differ  in  the  quantity  of  antitoxin  pro- 
duced. Similar  differences  are  observed  in  the  smaller  animals. 

Immunization  with  a  single  antigen  usually  produces  several  differ- 
ent antibodies,  although  for  diagnostic  or  therapeutic  purposes  one 
usually  predominates.  Thus  immunization  of  a  rabbit  with  dead 
typhoid  bacilli  produces  agglutinin,  opsonin,  bacteriolysin,  and  comple- 
ment-fixing bodies,  although  the  agglutinin  is  probably  the  most  prom- 
inent and  is  used  in  diagnosis.  Immunization  with  the  diphtheria 
bacillus  and  its  toxin  leads  to  the  formation  of  an  antitoxin,  opsonin, 
and  complement-fixing  body,  although  antitoxin  is  by  far  the  most 
prominent  and  is  used  therapeutically. 

We  give  here  methods  for  making  various  immune  serums  from  small 
animals,  to  be  used  for  the  purpose  of  study  and  for  aiding  diagnosis. 
Curative  serums,  such  as  diphtheria  and  tetanus  antitoxin,  antimeningo- 
coccus  serum,  etc.,  are  made  on  a  larger  scale  by  immunizing  horses. 


GENERAL  TECHNIC 

1.  The  antigens  are  usually  injected  subcutaneously,  intramuscularly, 
intraperitoneally,  or  intravenously.  As  a  rule,  the  sooner  the  antigen 
comes  in  relation  with  the  body-cells,  the  more  rapid  is  the  immunity 
gained,  and  for  this  reason  the  intravenous  and  intraperitoneal  routes 
are  frequently  chosen. 


GENERAL    TECHNIC  67 

2.  No  fixed  rules  as  to  the  amount  to  be  injected  can  be  given. 
Experience  may  show  a  general  method  to  be  the  most  successful,  but 
as  previously  mentioned,  the  general  condition  and  reaction  of  the  ani- 
mal will  be  the  main  guide  as  to  the  amount  and  frequency  of  the  in- 
jections.    Severe  reactions  may  yield  unsuccessful  results,  and  doses 
so  small  as  apparently  to  give  no  reaction  may  lead  to  a  high-grade  im- 
munity. 

3.  A  single  injection  seldom  yields  a  highly  valent  serum.     Re- 
peated inoculations  are  usually  necessary,  and  may  be  given  in  the  fol- 
lowing way: 

(a)  A  small  dose  of  antigen  is  injected.  If  a  reaction  sets  in,  wait 
until  this  has  subsided,  and  then,  after  the  fifth  day,  make  a  second  in- 
jection of  a  somewhat  larger  dose.  After  another  interval  of  from  five 
to  seven  days  a  third  injection  of  a  still  larger  dosage  is  administered, 
and  so  on  for  two  or  more  injections. 

(6)  Same  as  preceding  method,  except  that  the  first  dose  is  the  max- 
imum one;  subsequent  injections  are  of  decreasing  amounts. 

(c)  Same  as  preceding,  with  a  constant  dose  of  antigen  at  each  in- 
jection which  does  not  produce  a  severe  reaction. 

(d)  For  several  successive  days  a  small  or  medium-sized  dose  of 
antigen  is  injected  (Gay). 

The  first  three  methods  give  excellent  results,  and  the  third  is  es- 
pecially useful  when  a  serum  is  needed  as  soon  as  possible. 

4.  It  must  be  emphasized  that  good  results  are  largely  dependent  on 
the  care  with  which  the  animals  are  injected.     The  operator  should  work 
as  aseptically  as  possible,  especially  when  giving  intraperitoneal  and 
intravenous  injections,  and  avoid  the  production  of  embolism  by  the 
injection  of  air  or  solid  particles.     Give  the  injections  slowly,  and  take 
particular  care  of  the  animals.     However  carefully  the  injections  may 
be  given,  unsatisfactory  results  not  infrequently  occur.     Either  the 
animal  refuses  to  react  with  the  production  of  antibodies,  or  dies  just 
when  immunization  is  about  completed.     It  is,  therefore,  good  practice 
to  immunize  more  than  one  rabbit  at  one  sitting,  in  order  that  immune 
serum  may  be  had  at  the  time  planned. 

Antigens. — Animals  may  be  actively  immunized  with  the  following 
substances : 

1.  With  soluble  bacterial  toxins,  as  in  the  manufacture  of  antitoxins. 

2.  With  bacteria  themselves;    whether  living,  attenuated,  or  dead, 
as  in  making  bacterial  agglutinins,  precipitins,  immune  opsonins,  and 
lysins  (bacteriolysins) . 


68    METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF  ANIMALS 

3.  With  soluble  alien  proteins,  as  serum,  milk,  egg-albumen,  etc., 
as  in  the  manufacture  of  precipitins. 

4.  With  various  alien  cells,  as  erythrocytes,  kidney  cells,  sperma- 
tozoa, etc.,  in  the  manufacture  of  cytotoxic  serums,  such  as  hemolysins, 
nephrotoxin,  spermatotoxin,  etc. 

PRODUCTION  OF  ANTITOXIN 

Diphtheria  and  tetanus  antitoxins  are  prepared  on  a  large  scale  by 
the  inoculation  of  horses  with  increasing  doses  of  the  respective  toxins. 

The  methods  for  preparing  these  antitoxins,  and  also  of  antimeningo- 
coccus,  antistreptococcus,  antipneumococcus,  and  other  curative  se- 
rums are  given  in  the  chapter  on  Antitoxins  and  Passive  Immunization. 

PRODUCTION  OF  AGGLUTININS 

Agglutinating  serums  are  frequently  of  much  value  in  making  a 
bacteriologic  diagnosis  of  typhoid  fever  and  cholera.  For  this  purpose 
unknown  microorganisms  are  mixed  with  proper  dilutions  of  known 
immune  serum,  and  the  presence  or  the  absence  of  agglutination  noted. 
As  a  rule,  rabbits  are  used  in  the  preparation  of  these  serums;  for  the 
production  of  larger  quantities  of  serum,  goats  and  horses  are  occasionally 
employed. 

The  injections  may  be  given  intravenously  or  intraperitoneally; 
occasionally  the  first  injections  are  given  subcutaneously,  to  be  followed 
later  by  intraperitoneal  injections.  By  heating  the  cultures  at  a  temper- 
ature not  exceeding  60°  C.  for  from  one-half  to  one  hour,  there  is  less 
danger  in  the  subsequent  handling  of  the  cultures  and  agglutinins  are 
readily  produced. 

1.  Use   forty-eight-hour   agar   cultures   of  the   organism,   such   as 
Bacillus  typhosus,  Spirillum  cholerse,  etc.     Bouillon  cultures  may  be 
used,  but  are  not  recommended  on  account  of  the  various  other  con- 
stituents present  in  the  medium. 

2.  With  a  sterilized  three-millimeter  platinum  loop  remove  one 
loopful  of  culture  and  rub  up  in  2  c.c.  of  sterile  salt  solution  in  a  small 
test-tube  until  a  homogeneous  emulsion  is  secured. 

3.  Heat  the  emulsion  for  thirty  minutes  at  60°  C.  in  a  water-bath. 

4.  Inject  intravenously. 

5.  Give  four  more  injections  at  intervals  of  a  week  as  follows: 

Second  dose:  2  loopfuls  in  2  c.c.  NaCl  solution,  heated. 
Third  dose:  4  loopfuls  in  2  c.c.  NaCl  solution,  heated. 
Fourth  dose:  6  loopfuls  in  2  c.c.  NaCl  solution,  heated. 
Fifth  dose:    1  agar  slant  in  4  c.c.  NaCl  solution,  heated. 


PRODUCTION    OF   BACTERIOLYSINS    (BACTERIOLYTIC   SERUM)    69 

6.  One  week  after  the  last  injection  has  been  made  the  blood  is 
tested,  and  if  found  of  satisfactory  titer,  the  animal  is  killed  and  the 
serum  secured. 

Intraperitoneal  Method  (Rabbit). — 1.  Same  as  the  preceding,  ex- 
cepting that  larger  doses  are  given. 

First  dose:  2  loopfuls  in  4  c.c.  NaCl  solution,  heated. 
Second  dose:  4  loopfuls  in  4  c.c.  NaCl  solution,  heated. 
Third  dose:  6  loopfuls  in  4  c.c.  NaCl  solution,  heated. 
Fourth  dose:  1  agar  slant  in  5  c.c.  NaCl  solution,  heated. 
Fifth  dose:  1  agar  slant  in  5  c.c.  NaCl  solution,  heated. 
2.  The  blood  is  tested  one  week  after  the  last  injection  has  been 
made. 

PRODUCTION  OF  IMMUNE  OPSONINS 

1.  These  may  be  produced  in  the  same  manner  as  the  agglutinating 
serums,  immune  opsonins  being  readily  demonstrated  in  the  same  se- 
rums.    For  actual  diagnostic  work,  artificial  immune  opsonins  are  sel- 
dom required,  but  to  secure  an  immune  serum  for  experimental  studies 
on  opsonins  a  culture  of -Staphylococcus  pyogenes  aureus  may  be  used  in 
immunizing  a  guinea-pig  as  follows: 

First  dose:  1  loopful  of  twenty-four-hour  agar  culture  in  2  c.c. 

NaCl  solution  heated  for  one-half  hour  at  58°C.  and  given 

subcutaneously. 
Second   dose:    1    loopful    in   2  c.c.   NaCl,   heated;    intraperi- 

toneally. 

Third  dose:  2  loopfuls  in  2  c.c.  NaCl,  heated;  intraperitoneally. 
Fourth  dose:  3  loopfuls  in  2  c.c.  NaCl,  heated;  intraperitoneally. 
Fifth  dose:  6  loopfuls  in  2  c.c. "NaCl,  heated;  intraperitoneally. 

2.  Bleed  the  animals  one  week  after  the  last  injection  has  been  made. 

3.  Owing  to  its  large  size,  Bacillus  anthracis  may  be  substituted. 
This  is  a  spore-forming  organism,  and  since  it  is  dangerous  unless  scru- 
pulous care  in  handling  is  exercised,  it  is  not  usually  wise  to  employ  it  in 
experimental  work. 

PRODUCTION   OF  BACTERIOLYSINS   (BACTERIOLYTIC   SERUM) 
1.  These  are  prepared  in  exactly  the  same  manner  as  agglutinins. 
In  practical  diagnostic  work  the  Spirillum  cholera?  is  most  frequently 
used.     In  experimental  studies  of  bacteriolysis  the  typhoid  bacillus 
and  its  immune  serum  may  be  employed  with  equal  success. 


70   METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF  ANIMALS 

PRODUCTION  OF  PREOPITINS 

Precipitin  immune  serums  are  frequently  of  value  in  making  a  differ- 
entiation of  the  proteids,  as  in  the  examination  of  blood-stains,  meat, 
milk,  cheese,  etc.  They  are  usually  prepared  by  immunizing  large 
rabbits  with  injections  of  the  sterile  antigen.  Injections  may  be  given 
intravenously  or  intraperitoneally,  the  former  usually  yielding  the  more 
potent  serums. 

Any  foreign  serum  may  be  used  in  the  preparation  of  precipitins, 
such  as  that  of  the  human,  horse,  ox,  dog,  cat,  guinea-pig,  etc.  To  pro- 
duce an  antirabbit  precipitin  a  guinea-pig  is  immunized  by  intraperi- 
toneal  injections  of  rabbit  serum.  An  antihuman  precipitin  serum  of 
high  titer  is  usually  obtained  with  difficulty.  It  is  good  practice  to 
immunize  a  number  of  rabbits  with  each  antigen,  as  some  animals  will 
produce  no  precipitin  whatever  the  method  used. 

In  preparing  precipitins  for  the  purpose  of  identifying  blood-stains 
whole  blood  may  be  injected.  It  is  better,  however,  to  use  serum  only, 
as  the  immune  serum  may  be  used  in  diagnosis,  according  to  the  method 
of  complement  fixation,  when  the  presence  of  hernolysin  is  not  advisable. 

Serum  Precipitins  (Intravenous  Method). — First  Method. — Three 
injections  are  given — of  5,  10,  and  15  c.c. — on  each  of  three  successive 
days,  and  the  animals  are  bled  twelve  days  after  the  last  injection  has 
been  made. 

Second  Method. — One  injection  of  30  c.c.  of  serum  may  be  given,  and 
followed  twelve  days  later  by  bleeding. 

Third  Method. — A  slower  method  consists  in  giving  the  injections  at 
intervals  of  five  days.  After  the  third  dose  a  few  cubic  centimeters  of 
blood  are  withdrawn  from  the  ear,  and  the  serum  titrated,  as  rabbits  are 
most  prone  to  succumb  after  the  third  dose,  and  in  many  instances  the 
serum  is  of  such  strength  as  to  require  no  further  immunization.  The  ani- 
mals are  bled  one  week  after  the  last  injection  has  been  given. 

Doses  may  be  given  as  follows : 

First  dose:  10  c.c.  serum  intravenously. 
Second  dose:     8  c.c.  serum  intravenously. 
Third  dose:     5  c.c.  serum  intravenously. 
Fourth  dose:     5  c.c.  serum  intravenously. 
Fifth  dose:     3  c.c.  serum  intravenously. 
Sixth  dose:     3  c.c.  serum  intravenously. 

Fourth  Method. — Rabbits  may  be  immunized  by  making  intra- 
peritoneal  injections  after  any  of  the  foregoing  methods,  and  with  the 
same  or  slightly  larger  doses. 


PRODUCTION   OF   HEMOLYSINS  71 

Milk  Precipitins  (Lactoserums). — These  are  prepared  by  immuniz- 
ing large  rabbits  with  intravenous  or  intraperitoneal  injections  of  milk, 
that  of  either  the  human  or  the  lower  animals.  Rabbits  should  be  im- 
munized with  at  least  two  kinds  of  milk  in  order  to  obtain  different 
lactoserums  for  the  study  of  specificity.  The  milk  used  for  the  injec- 
tions should  be  as  sterile  as  possible,  and  if  heated  to  56°  C.  for  one-half 
hour  before  the  injections  are  made,  the  protein  remains  unchanged  and 
the  rabbits  are  less  likely  to  succumb.  Animals  may  be  immunized 
in  the  same  manner  and  with  the  same  sized  doses  as  were  directed  for 
the  preparation  of  serum  precipitins. 

Bacterial  Precipitins. — It  is  usually  customary  to  differentiate  be- 
tween the  bacterial  and  the  protein  precipitins,  but  for  practical  pur- 
poses this  division  is  superfluous,  as  the  bacterial  precipitins  are  simply 
antiserums  prepared  by  immunization  with  bacterial  protein. 

PRODUCTION  OF  HEMOLYSINS 

Because  of  their  use  in  the  serum  diagnosis  of  syphilis  and  other  in- 
fections, hemolysins  possess  great  practical  value.  They  are  best  pro- 
duced by  injecting  rabbits  with  washed  human  or  sheep  erythrocytes, 
or  with  those  of  some  animal  of  another  species.  Rabbits  differ  con- 
siderably in  their  power  to  form  hemolysins,  and  for  some  unknown 
reason  hemolysins  are  more  readily  produced  with  some  erythrocytes 
than  with  others.  It  is  not  possible,  however,  to  immunize  every  kind 
of  animal  against  every  type  of  erythrocyte.  As  a  general  rule,  an 
animal  produces  a  better  hemolysin  the  more  remote  its  relationship  is 
to  the  animal  from  which  the  erythrocytes  for  making  the  injection  are 
taken. 

Hemolysins  may  be  prepared  as  the  result  either  of  intraperitoneal 
or  of  intravenous  injections  of  erythrocytes  washed  at  least  three  or 
four  times  to  remove  all  traces  of  serum.  Unless  an  antihuman  hemoly- 
sin is  required  within  a  short  space  of  time,  better  results  are  obtained, 
as  a  rule,  by  using  the  slower,  intraperitoneal  method. 

In  immunizing  rabbits  it  must  be  remembered  that  the  quantity 
of  amboceptor  produced  bears  no  direct  relation  to  the  size  of  the  doses 
given.  Thus,  a  highly  potent  hemolytic  serum  may  be  prepared  by 
three  intravenous  injections  of  from  3  to  5  c.c.  of  a  10  per  cent,  suspen- 
sion of  washed  sheep  cells. 

It  must  be  emphasized  that  as  far  as  possible  an  aseptic  technic 
should  be  employed,  and  that  corpuscles  used  for  making  intravenous 


72   METHODS  FOR  EFFECTING  ACTIVE  IMMUNIZATION  OF  ANIMALS 

injections  should  be  filtered  to  remove  small  particles  of  fibrin,  and  pref- 
erably washed  four  times  with  sterile  salt  solution.  The  method  of 
preparing  corpuscles  for  injection  has  been  given  in  a  preceding  chapter. 
After  the  third  or  fourth  injection  the  animal  should  be  bled  from  the 
ear  and  the  serum  tested,  as  animals  not  infrequently  succumb  after 
the  fourth  injection,  and  many  possess  serums  of  high  potency  after  re- 
ceiving three  injections.  By  this  method  success  is  better  assured. 

Intravenous  Method. — For  the  preparation  of  most  hemolysins 
the  following  methods  yield  very  good  results.  Antihuman  hemolysin 
is  more  difficult  to  prepare,  and  I  have  generally  found  that  a  slower, 
intraperitoneal  method  will  yield  better  results. 

First  Method. — Four  injections  of  5  c.c.  each  of  a  10  per  cent,  sus- 
pension of  washed  cells  at  intervals  of  three  days.  The  animal  is  bled 
three  or  four  days  after  receiving  the  last  injection. 

Second  Method.— Three  injections  of  10  c.c.  each  of  a  10  per  cent, 
suspension  of  washed  cells  on  each  of  three  successive  days.  The  rab- 
bit is  bled  four  days  after  receiving  the  last  injection  (after  the  method  of 
Gay).  Instead  of  these  large  injections,  1  c.c.  of  corpuscles,  removed 
after  thorough  centrifugalization  and  diluted  with  sufficient  sterile 
salt  solution,  may  be  given  on  each  of  three  successive  days. 

Third  Method. — A  slower  method  consists  in  making  the  injections  of 
a  suspension  of  corpuscles  every  five  days.  The  cells  must  be  thoroughly 
washed  to  free  them  from  all  traces  of  serum;  if  this  is  not  done,  the 
animals  may  die  of  anaphylaxis  during  the  course  of  immunization; 
animals  should  be  tested  after  the  third  dose  has  been  injected: 

First  dose:     3  c.c.  of  a  10  per  cent,  suspension  of  corpuscles. 
Second  dose:     5  c.c.  of  a  10  per  cent,  suspension  of  corpuscles. 
Third  dose:  10  c.c.  of  a  10  per  cent,  suspension  of  corpuscles. 
Fourth  dose:  15  c.c.  of  a  10  per  cent,  suspension  of  corpuscles. 
Fifth  dose:  20  c.c.  of  a  10  per  cent,  suspension  of  corpuscles. 

Fourth  Method. — In  the  preparation  of  antihuman  amboceptor, 
Noguchi  advises  giving  four  intravenous  injections  of  washed  cells, — 4  c.c., 
3  c.c.,  4  c.c.,  3  c.c., — and  possibly  another — 4  c.c. — at  intervals  of  four  or 
five  days;  these  amounts  of  packed  cells  are  suspended  in  10  c.c. of  sterile 
salt  solution.  Rabbits  are  bled  one  week  after  the  last  injection  is  given. 

Intraperitoneal  Method. — In  this  method  the  most  careful  aseptic 
precautions  should  be  observed  in  washing  the  cells  and  in  giving  the 
injections,  or  the  animals  are  likely  to  succumb  from  peritonitis  just 
about  the  time  they  are  fully  immunized  and  ready  for  bleeding.  In- 
jections may  be  given  every  four  or  five  days,  and  one  week  after  re- 
ceiving the  last  injection  the  rabbits  are  to  be  bled. 


PRODUCTION  OF  CYTOTOXINS  73 

This  method  is  especially  serviceable  in  preparing  antihuman 
amboceptor.  Whenever  possible,  blood  should  be  collected  aseptically, 
and  the  corpuscles  washed  just  before  the  injections  are  given.  Pla- 
cental  blood  is  likely  to  be  infected  and  hemolyzed,  and  frequently 
yields  unsatisfactory  results.  In  preparing  the  antihuman  amboceptor 
several  rabbits  should  be  immunized  at  the  same  time,  the  doses  being: 

First  dose:     5  c.c.  of  the  washed  corpuscles. 

Second  dose:    8  c.c.  of  the  washed  corpuscles. 

Third  dose:  12  c.c.  of  the  washed  corpuscles. 

Fourth  dose:  15  c.c.  of  the  washed  corpuscles. 

Fifth  dose :  20  c.c.  of  the  washed  corpuscles. 

A  combined  intravenous-intraperitoneal  series  of  injections  is  fre- 
quently of  service  in  producing  a  highly  potent  antihuman  hemolytic 
serum.  Three  intravenous  injections  of  4  c.c.  each  of  washed  cells 
diluted  with  6  c.c.  of  sterile  salt  solution  are  given  every  five  days;  sub- 
sequent injections  are  made  by  giving  1  c.c.  of  corpuscles  intraperito- 
neally,  followed  one  hour  later  by  4  c.c.  intravenously.  A  series  of  three 
or  four  of  these  combined  injections  are  made  at  intervals  of  five  days, 
the  animals  being  tested  at  frequent  intervals  to  determine  the  hemolytic 
strength  of  the  serum. 

PRODUCTION  OF  CYTOTOXINS 

The  term  "cytotoxins"  is  usually  applied  to  cell  toxins  other  than 
hemolysins,  such  as  nephrotoxins,  spermatotoxins,  etc.,  and  although 
"lysin"  is  frequently  used,  the  term  " toxin"  is  better,  being  descriptive 
of  the  changes  produced  by  all  cell  toxins  except  the  hemolysins  and 
the  bacteriolysins. 

Cytotoxic  serums  can  be  made  theoretically  for  any  cell,  but  only 
the  hemolysins  possess  much  practical  value.  The  cytotoxins  are 
prepared  with  some  difficulty  by  injecting  emulsions  of  cells  from  one 
animal  into  another.  Immunization  should  always  be  conducted  by 
means  of  intraperitoneal  or  subcutaneous  injection. 

For  the  purpose  of  studying  the  action  of  cytotoxic  serum,  nephro- 
toxic  serum  is  preferably  to  be  used,  as  the  effects,  e.  g.,  the  production 
of  albuminuria,  may  be  observed.  A  series  of  two  or  three  animals 
should  be  carried  along  at  the  same  time,  as  many  die  after  the  third 
injection.  So  far  as  possible  an  aseptic  technic  should  be  carried  out. 

Practically  all  cytotoxic  serums  are  hemolytic  partly  because  of  the 
great  difficulty  of  removing  all  traces  of  blood  from  the  organ  used  in 


74     METHODS    FOR    EFFECTING    ACTIVE    IMMUNIZATION    OF    ANIMALS 

preparing  the  emulsion.  This  difficulty  may  be  reduced  to  a  minimum 
by  thoroughly  washing  the  organ  or  tissue  prior  to  preparing  an  emulsion 
for  injection. 

The  following  method,  after  Pearce,  illustrates  the  mode  of  preparing 
a  nephrotoxic  serum  by  immunizing  rabbits  with  dog  kidney. 

1.  Anesthetize  a  dog  with  ether,  open  the  abdomen,  wash  the  blood 
out  of  the  kidneys  by  inserting  a  cannula  high  up  in  the  abdominal  aorta, 
and  flushing  with  from  six  to  ten  liters  of  salt  solution;  open  the  vena 
cava. 

2.  Remove  the  kidney  as  aseptically  as  possible,  and  grind  it  in  a 
meat-grinder;  rub  through  fine-meshed  wire  gauze,  and  wash  the  residue 
in  several  changes  of  sterile  salt  solution.     The  fat  should  be  rejected 
and  only  the  cortex  used. 

3.  Weigh  and  suspend  10  grams  of  the  substance  in  30  c.c.  of  sterile 
salt  solution. 

4.  Inject  the  rabbit  intraperitoneally  with  10  c.c.  of  the  emulsion 
every  seven  days. 

5.  After  the  third  dose  has  been  given  test  the  serum,  as  subsequent 
doses  increase  the  danger  of  losing  the  animal. 

6.  The  animal  is  bled  one  week  after  receiving  the  last  injection. 

7.  The  injection  of  this  serum  into  dogs  is  usually  followed  by  al- 
buminuria  and  possibly  hemoglobinuria.     This  subject  is  further  con- 
sidered under  the  head  of  Practical  Exercises  with  Cytotoxins. 

Some  investigators  have  asserted  that  by  effecting  immunization 
with  the  nucleoproteids  of  an  organ  more  specific  cytotoxic  serums  are 
secured.  These  claims  have  not  been  confirmed  by  Pearce,  Wells, 
and  others.  (See  Chapter  XXV.) 

Nucleoproteins  may  be  secured  as  follows:  Grind  the  organ  or  tissue  in  a  meat- 
grinder,  and  finally  rub  up  with  sand  with  a  pestle  in  a  mortar;  add  two  volumes  of 
normal  salt  solution,  and  pass  through  a  meat  press;  collect  the  effluent,  place  in  a 
refrigerator  for  twenty-four  hours  and  then  filter  through  gauze  and  centrifuge  the 
filtrate;  to  the  supernatant  fluid  add  acetic  acid  to  remove  the  nucleoproteins.  Place 
hi  the  refrigerator  for  eighteen  hours  and  centrifuge.  Collect  the  sediment  and  wash 
several  times  with  normal  salt  solution.  Dissolve  the  sediment  in  normal  salt 
solution  containing  0.5  per  cent,  sodium  carbonate.  Reprecipitate  with  acetic  acid, 
wash,  and  redissolve  in  the  alkaline  solution. 


CHAPTER  V 
THE  PRESERVATION  OF  SERUMS— METHODS 

IT  is  well  to  remember  that  serum  collected  shortly  after  a  meal  is 
likely  to  be  cloudy  or  opalescent;  it  is  therefore  advisable  that  blood 
be  collected  several  hours  after  eating  or  during  a  period  of  fasting. 

After  securing  a  specimen  of  blood,  the  container  should  be  set  aside 
and  kept  at  room  temperature  until  the  serum  separates.  If  the  serum 
is  to  be  used  at  once,  blood  may  be  collected  in  centrifuge  tubes,  allowed 
to  coagulate,  and  then  broken  up,  as  gently  as  possible,  with  a  sterile 
glass  rod  and  thoroughly  centrifuged.  On  account  of  the  mechanical 
rupture  of  erythrocytes,  such  serums  are  usually  tinged  with  hemo- 
globin. After  serum  has  separated  from  the  clot  it  should  be  trans- 
ferred to  another  tube,  or,  if  this  is  not  immediately  possible,  the  con- 
tainer should  be  placed  in  the  refrigerator,  to  retard  hemolysis,  which 
may  soon  occur  and  render  the  serum  unfit  for  many  purposes. 

Small  amounts  of  serum  are  best  removed  from  the  clot  with  a  cap- 
illary pipet  and  teat,  or  with  an  ordinary  graduated  pipet  with  rubber 
tubing  and  mouth-piece,  in  order  that  one  may  see  exactly  what  he  is 
doing  and  not  disturb  the  clot.  As  a  perfectly  clear  serum  is  always  to  be 
desired,  serums  mixed  with  corpuscles  should  be  centrifuged. 

It  may  be  stated,  as  a  general  rule,  that  all  normal  and  immune  se- 
rums should  be  collected  as  aseptically  as  possible,  and  handled  in  a 
careful  and  aseptic  manner,  so  as  to  insure  a  clear  and  sterile  product. 
Notwithstanding  the  method  of  preservation  all  serums  should  be  kept 
in  a  refrigerator  or  ice-chest  at  a  low  temperature. 

PRESERVATION  OF  NORMAL  SERUMS 

Normal  serums  that  are  to  be  used  for  purposes  of  immunization  are 
best  preserved  in  small  amounts  in  separate  ampules,  or  in  a  large  stock 
bottle  holding  from  100  to  200  c.c.  and  well  stoppered.  In  the  produc- 
tion of  precipitin-serum,  for  example,  sufficient  serum  of  an  animal  may 
be  obtained  at  a  single  sitting  for  the  whole  course  of  injections,  and  this 
serum  is  best  preserved  in  separate  ampules.  Each  ampule  should 
contain  sufficient  serum  for  one  injection,  and  be  sealed  and  marked. 

75 


76  THE    PRESERVATION    OF   SERUMS — METHODS 

In  this  manner  the  risk  of  contaminating  a  stock  bottle  is  obviated. 
In  the  preservation  of  normal  serum  or  the  serum  of  luetics  to  be  used 
as  controls  for  the  Wassermann  reaction,  it  is  better  to  store  them  in 
small  amounts  in  sterile  ampules. 

As  a  rule,  it  is  best  not  to  add  a  preservative  to  serums  that  are  to  be 
used  for  purposes  of  immunization,  for  if  the  dose  of  serum  is  large, 
enough  preservative  may  be  injected  to  place  the  health  of  the  animal 
in  jeopardy.  However,  chloroform  may  be  added  in  proportion  of  1:10 
or  1 : 20,  provided  the  serum  is  placed  in  the  incubator  or  heated  in  the 
water-bath  at  40°  C.  for  fifteen  minutes  in  order  to  drive  off  the  chlo- 
roform previous  to  injection. 


PRESERVATION  OF  IMMUNE  SERUMS 

Immune  serums  may  be  preserved  either  in  the  fluid  or  in  the  dry 
form. 

Preservation  in  Fluid  Form  with  Antiseptics. — Practically  any 
immune  serum  may  be  preserved  in  the  fluid  state  by  adding  a  suit- 
able preservative  in  the  proper  dose  without  exerting  any  deleterious 
influence  on  the  antibody  content.  The  exceptions  to  this  general  rule 
are  the  precipitin-serums,  because  these  should  be  crystal  clear,  and 
a  preservative  may  render  the  serum  slightly  cloudy.  According  to 
Uhlenhuth,  Weidanz,  and  Wedemann,  such  serums  should  be  filtered 
through  a  'sterile  Berkefeld  filter  and  then  stored  without  adding  an 
antiseptic. 

Various  antiseptics  have  been  advocated  for  the  preservation  of 
serums.  Hemolytic  serum  is  well  preserved  by  adding  an  equal  amount 
of  chemically  pure  glycerin  to  the  serum  after  it  has  been  inactivated  by 
heating  at  55°  C.  for  a  half-hour  in  a  water-bath.  The  addition  of  0.1 
c.c.  of  a  1  per  cent,  solution  of  phenol  in  salt  solution  to  each  cubic 
centimeter  of  immune  serum  usually  suffices  to  keep  the  fluid  free  from 
contamination,  and  produces  only  very  slight,  if  any,  clouding.  Like- 
wise, the  addition  of  2  per  cent,  formalin  in  a  5  per  cent,  solution  of 
glycerin  in  normal  salt  solution,  in  the  proportion  of  1 : 10,  makes  a  very 
useful  antiseptic.  Neither  lysol  nor  trikresol  should  be  used  in  the 
preservation  of  a  serum,  as  they  are  more  likely  to  produce  clouding 
than  does  phenol. 

Preservation  in  Fluid  Form,  by  Bacteria-free  Filtration. —  If  serums 
are  to  be  preserved  in  fluid  form  without  the  addition  of  an  antiseptic, 
special  precautions  in  bleeding,  collecting,  and  separating  should  be 


PRESERVATION    OF   IMMUNE    SERUMS 


77 


observed.  If  contamination  has  probably  occurred,  the  serum  should 
be  filtered  through  a  sterile  Berkefeld  filter  (Fig.  34).  The  apparatus 
devised  by  Uhlenhuth  (see  Fig.  91)  is  especially  useful,  as  the  serum  is 
collected  at  once  in  a  sterile  container,  which  is  then  plugged  with  sterile 
cotton  and  placed  in  the  incubator  for  twenty-four  hours.  If  the  serum 


FIG.  34. — A  SMALL  BERKEFELD  FILTER. 

The  fluid  to  be  filtered  is  poured  into  the  glass  cylinder  surrounding  the  earthen 
or  porcelain  "candle."  Negative  pressure  within  the  candle  is  produced  by  the 
water-pump,  which  exhausts  the  air  from  the  flask.  The  nitrate  is  collected  in  the 
test-tube  within  the  filter  flask.  All  parts  are  readily  sterilized  in  an  Arnold  sterili- 
zer or  autoclave.  The  sterile  cotton  plug  prevents  air  contamination. 

proves  to  be  sterile,  it  is  transferred,  with  the  aid  of  a  sterile  pipet,  into 
ampules  of  1  c.c.  capacity.  These  are  sealed  hermetically  and  kept  in 
the  refrigerator. 

Small  amounts  of  serum  may  be  lost  in  a  large  filter,  and  a  smaller 
apparatus  should  therefore  be  used.  The  filter  shown  in  the  accom- 


78 


THE    PRESERVATION    OF   SERUMS — METHODS 


panying  illustration  (Fig.  35)  is  quite  serviceable,  as  the  flask  and 
earthen  candle-filter  may  be  wrapped  in  a  towel  and  sterilized  in  the 
autoclave.  The  apparatus  may  carefully  be  attached  to  a  suction 
pump,  and  the  serum  pipeted  off  into  the  hollow  of  the  candle  and  fil- 
tered, the  filtrate  being  removed,  at  the  completion  of  the  process,  by 
another  sterile  pipet. 


FIG.  35. — A  FILTER. 

The  cotton  plug  in  the  " candle"  is  removed  and  the  fluid  poured  within  the 
candle  (hollow).  The  water  is  then  turned  on  and  the  stop-cocks  are  opened;  a 
vacuum  is  produced  within  the  flasks,  which  draws  the  fluid  through  the  candle. 
The  filter  is  readily  cleansed  and  sterilized  (autoclave)  and  is  quite  efficient. 

Care  should  be  exercised  regarding  the  reaction  of  Berkefeld  filters 
and  particularly  new  filters.  Before  use  they  should  be  boiled  in  distilled 
water  at  least  three  times  for  five  minutes  each  time,  and  scrubbed  with 
a  small  soft  brush  after  each  boiling.  After  the  filter  is  set  up,  hot, 
neutral,  distilled  water  should  stand  in  it  for  about  five  minutes  and  then 


PRESERVATION    OF    IMMUNE    SERUMS  79 

washed  through  under  gentle  pressure  until  the  fluid  is  clear  and  neutral 
to  phenolphthalein,  when  the  filter  is  ready  for  use.  After  use,  the  filter 
should  be  boiled  in  distilled  water,  scrubbed,  and  dried  in  the  air. 

Preservation  in  Fluid  Form,  by  Freezing. — Freezing  a  serum  often 
renders  it  cloudy  or  causes  a  precipitate  to  be  deposited,  and  interferes 
with  the  usefulness  of  a  serum  that  should  be  absolutely  clear.  Freezing 
is  the  only  practicable  method  so  far  devised  for  the  preservation  of 
thermolabile  substances,  such  as  complement.  A  small  apparatus, 
named  the  "Frigo,"  has  been  devised  for  this  purpose  by  Morgenroth. 
A  satisfactory  apparatus  may  be  made  by  constructing  a  wooden  box 
with  a  smaller  sheet-metal-covered  inner  compartment,  the  space  be- 
tween them  being  well  packed  with  sawdust.  This  inner  box  is  then 
filled  with  crushed  ice,  and  the  whole  is  covered  with  a  lid  lined  with 
several  layers  of  felt. 

Preservation  in  Powder  Form. — When  serum  is  poured  out  in  thin 
layers  and  dried,  it  forms  yellowish,  amorphous  masses,  that  may  be 
collected  and  ground  into  a  powder,  which  keeps  well  and  forms  an 
excellent  medium  for  the  preservation  of  many  immune  serums,  espe- 
cially those  of  the  agglutinating  type.  Various  toxins,  such  as  tetanus 
toxin  and  cobra  venom,  may  also  be  preserved  in  this  form. 

The  serum  or  toxin  may  be  spread  out  in  thin  layers  on  large  glass 
plates,  or  placed  in  shallow  dishes  and  dried  in  the  incubator.  After  a 
few  hours  the  dried  serum,  which  adheres  only  slightly  to  the  dish,  can 
be  removed  with  a  spatula  and  placed  in  a  mortar,  and  ground  and 
stored  in  sealed  tubes. 

The  drying  process  is  better  carried  out  in  vacuo,  and  the  large  serum 
institutes  are  provided  with  these  special  drying  apparatus.  A  simple 
form  may  be  prepared  after  the  method  of  Taeze,  as  follows:  Place  a 
large  glass  bell-jar  with  a  ground  base  and  a  large  opening  at  the  top  on 
a  polished  iron  plate.  Set  this  on  a  large  tripod,  as  this  will  facilitate 
heating  with  a  Bunsen  burner.  The  serum  is  placed  within  the  jar  in 
a  shallow  dish,  and  the  jar  fastened  to  the  iron  plate  with  hot  paraffin 
or  wax.  The  opening  at  the  top  is  closed  with  a  three-holed  rubber 
stopper:  one  hole  carries  a  thermometer;  a  second  is  connected  with  a 
manometer  (not  absolutely  necessary),  and  the  third  carries  a  bent  glass 
tube  which  is  connected,  by  means  of  thick-walled  rubber  tubing,  to  a 
suction  pump.  A  low  flame  is  kept  burning  so  as  to  keep  the  tempera- 
ture at  about  35°  C.  The  degree  of  vacuum  secured  makes  little  differ- 
ence, and  usually  that  obtained  with  an  ordinary  water-suction  pump, 
allowing  for  leaks  in  the  tubing,  is  sufficient,  rendering  manometric 
measurements  unnecessary. 


80  THE    PRESERVATION    OF    SERUMS METHODS 

I  have  secured  equally  good  results  in  evaporating  sera  and  tissue 
extracts  with  an  ordinary  electric  fan  enclosed  in  a  properly  sized  oblong 
wooden  box  to  concentrate  the  air  current. 

The  dried  serum  should  be  dissolved  in  sterile  normal  salt  solution 
before  it  is  used. 

Preservation  in  Dried  Paper  Form. — This  is  a  very  serviceable 
method  for  preserving  hemolysins,  and  to  a  lesser  extent,  agglutinins. 
In  the  preservation  of  hemolytic  amboceptor  Noguchi  advises  the  use 
of  Schleich  and  Schull's  paper  No.  597.  The  paper  is  cut  into  squares 
about  10  by  10  cm.,  and  saturated  with  the  serum  which,  after  prelimi- 
nary titration,  has  been  found  satisfactory.  Sufficient  serum  is  added  to 
wet  the  sheets  evenly,  any  excess  of  serum  being  absorbed  with  other 
sheets  of  paper.  Each  square  is  placed  separately  upon  a  clean  sheet  of 
unbleached  muslin  and  dried  at  room  temperature.  When  thoroughly 
dry,  the  squares  are  carefully  ruled  off  with  a  hard  pencil  into  widths  of 
about  5  mm.,  and  cut  into  strips.  The  paper  is  then  standardized  and 
preserved  in  dark  glass  vials  in  a  cool,  dark  place. 

Preservation  in  the  Living  Animal. — In  the  living  animal  an  im- 
mune serum  may  be  preserved  by  removing  a  small  amount  of  blood 
from  time  to  time  as  needed,  the  titer  being  preserved  or  raised  by 
occasional  injections.  This  method,  however,  may  be  unsatisfactory 
and  expensive,  especially  with  the  smaller  animals,  as  they  frequently 
show  a  marked  tendency  to  sicken  and  die,  or  may  succumb  to  anaphy- 
laxis.  After  a  time,  too,  they  fail  to  respond  to  injections  with  the 
formation  of  antibodies,  a  condition  ascribed  to  atrophy  of  the  cell- 
receptors  (receptoric  atrophy). 


PART  II 

CHAPTER  VI 

INFECTION 

Infection  is  the  successful  invasion  and  growth  of  microorganisms  in 
the  tissues  of  the  body. 

The  skin  and  adjacent  mucous  membranes  contain  numerous  micro- 
organisms, and  under  normal  conditions  these  may  invade  the  tissues, 
but  they  are  usually  quickly  destroyed  and  unable  to  proliferate,  so  that 
mere  invasion  does  not  necessarily  constitute  infection. 

Unfortunately,  custom  has  sanctioned  the  use  of  the  term  infection 
as  synonymous  with  contamination.  The  bacteriologist  may  speak  of 
the  air,  water,  or  his  culture  medium  as  being  infected  when  they  con- 
tain microorganisms,  or,  in  other  words,  are  not  sterile;  similarly  the 
surgeon  may  speak  of  a  knife  or  splinter  of  wood  as  being  infected 
whereas,  while  these  may  be  infective  or  capable  of  producing  infection, 
it  is  more  accurate  to  speak  of  them  as  being  contaminated.  In  the 
early  days  of  bacteriology,  the  mere  presence  of  microorganisms  in  or 
on  the  skin  and  mucous  membranes  was  regarded  as  equivalent  to  in- 
fection. It  is  now  well  known  that  a  person  may  harbor  various  micro- 
organisms, such  as  staphylococci,  streptococci,  and  pneumococci,  with- 
out apparent  injury  to  the  host,  and  this  surface  contamination,  or  even 
occasional  invasion  of  the  tissues,  does  not  necessarily  indicate  that  the 
host  has  been,  is,  or  will  be  ill. 

Definition. — When,  however,  microorganisms  have  passed  the  normal 
barriers  of  the  skin  or  mucous  membranes  and  have  invaded  and  prolifer- 
ated in  the  deeper  tissues,  the  process  is  spoken  of  as  an  infection. 

By  common  consent,  the  term  infestation,  or  infestment,  is  being 
applied  in  a  similar  manner  to  the  presence  and  growth  of  animal 
parasites;  thus  the  intestine  may  be  infected  with  Bacillus  typhosus  and 
infested  by  Taenia  saginata. 

A  microorganism  may  be  intimately  associated  with  and  have  its 
normal  habitat  in  a  certain  part  of  the  body  and  do  no  harm  until  special 
conditions  arise,  when  it  may  rapidly  invade  the  tissues  and  produce 
infection;  this  condition  has  been  described  by  Adami  as  a  subinfection, 
and  is  illustrated  by  the  constant  presence  of  staphylococci  and  strepto- 
6  81 


82  INFECTION 

cocci  in  the  tonsils  of  most  persons,  usually  harmless,  but  capable,  under 
special  conditions,  of  producing  severe  and  even  fatal  infection. 

The  abnormal  state  resulting  from  the  deleterious  local  and  general 
interaction  between  a  host  and  an  invading  bacterium,  with  consequent 
tissue  changes  and  symptoms,  constitutes  an  infectious  disease. 

As  has  previously  been  stated,  not  every  invasion  of  the  deeper 
tissues  by  microorganisms  results  in  injury  or  disease.  A  certain  num- 
ber of  bacteria  are  constantly  gaining  admission  to  the  deeper  tissues 
of  the  alimentary  and  the  respiratory  tracts,  without  producing  apparent 
injury  to  the  host,  as  they  tend  to  be  destroyed  very  soon  after  they 
gain  entrance.  Furthermore,  bacteriologic  studies  of  lymphatic  glands 
and  other  tissues  removed  during  life  or  soon  after  death  at  autopsy, 
not  infrequently  show  the  presence  of  diphtheroicl  bacilli  and  other 
microorganisms  possessing  feeble  or  no  demonstrable  pathogenic  powers 
and  indicating  that  various  bacteria  may  gain*  access  to  the  deeper 
tissues  without  producing  a  true  infection.  The  terms  invasion  and 
infection  are  not,  therefore,  synonymous.  Every  true  infection  is  ac- 
companied by  local  changes,  although  these  may  be  so  slight  as  to  escape 
notice;  an  infectious  disease  is  practically  made  up  of  similar  phenomena, 
but  these  are  of  an  exaggerated  or  marked  degree. 

The  hygienist  distinguishes  between — (1)  Sporadic,  or  isolated,  cases 
of  infection;  (2)  endemic,  in  which  a  certain  microbic  disease  affects  the 
inhabitants  of  a  given  area  year  after  year;  and  (3)  epidemic,  in  which  a 
disease  appears  suddenly  and  affects  a  large  number  of  inhabitants,  the 
number  of  cases  rapidly  increasing  and  decreasing.  Among  the  lower 
animals  equivalent  terms  for  the  types  just  described  are  sporadic, 
enzootic,  and  epizootic.  A  pandemic  disease  is  one  that  is  epidemic  over 
a  large  territory. 

In  all  infections  there  are  two  inseparable  factors  to  be  considered : 

1.  The  offensive  forces  of  the  infecting  agent,  dependent  upon  its 
pathogenic  or  disease-producing  nature  and  its  power  of  defending  itself 
against  the  antagonistic  forces  of  the  host  and  of  thriving  under  these 
conditions. 

2.  The  resistance  offered  by  the  host,  and  mainly  dependent  upon 
certain  physical  or  non-specific  local  factors  or  specific  antibodies,  which 
constitute  the  defensive  mechanism,  or  immunologic  factors. 

The  former  is  concerned  with  the  general  subject  of  infection,  and 
the  latter  with  that  of  immunity. 

Microorganisms  and  host  may  live  together  in  apparent  harmony, 
owing  to  the  ability  of  the  host  to  restrain  the  activity  of  the  micro- 
organism and  neutralize  its  injurious  effects  or  to  an  absence  of  infec- 


SOURCES   OF   INFECTION  83 

tivity  on  the  part  of  the  microorganism  until  the  vital  resistance  of  the 
host  is  diminished  or  the  pathogenicity  of- the  microorganism  is  increased, 
when  the  neutral  relations  are  disturbed  and  infection  occurs. 

RELATION  OF  INFECTION  TO  IMMUNITY 

From  what  has  been  said,  it  is  apparent  that  the  subject  of  infection 
forms  the  basis  for  the  study  of  immunology,  for,  paradoxic  as  it  would 
at  first  appear  to  be,  infection  must  usually  have  occurred  in  order  that 
immunity  may  be  acquired.  This  relation  is  not  always  apparent;  for 
instance,  man  and  some  of  the  lower  animals  may  possess  a  natural 
immunity  td  a  certain  parasite  because  of  the  presence  of  various  physi- 
cal or  non-specific  defensive  factors,  or  to  specific  antibodies  produced 
as  the  result  of  an  earlier  and  unrecognized  infection,  or  even  one  that 
has  been  inherited;  under  any  circumstances,  however,  natural  immu- 
nity is  usually  relative  and  seldom  absolute.  In  passive  immunity  the 
same  conditions  are  generally  operative,  and  the  antibodies  present  in 
the  serum  used  to  confer  a  passive  immunity  are  produced  in  some  other 
animal  as  the  result  of  an  active  infection. 

It  may  be  stated,  therefore,  that  specific  antibodies  are  produced 
only  by  stimulation  of  the  body-cells,  and  that  this  stimulation  is  fur- 
nished by  the  infecting  agent  either  in  living,  disease-producing  form,  or 
in  a  modified  and  attenuated  state,  i.  e.,  in  the  form  of  a  vaccine;  thus 
it  will  be  seen  that  infection  and  immunity  are  intimately  associated, 
and  that,  'generally  speaking,  there  can  be  no  pronounced  protection 
unless  infection  has  taken  place. 

SOURCES  OF  INFECTION 

Bacteria  are  to  be  found  everywhere.  For  general  purposes  they 
may  be  roughly  divided  into  two  classes — saprophytes  and  parasites. 
The  saprophytes  are  those  bacteria  which  thrive  best  in  dead  organic 
matter,  and  perform  the  very  important  function  of  reducing,  by  their 
physiologic  activities,  highly  organized  material  into  those  simple 
chemical  substances  that  may  again  be  utilized  by  the  plants  in  their 
constructive  processes,  and  in  this  manner  maintain  the  important 
chemical  relation  between  the  animal  and  the  plant  kingdom.  Para- 
sites, on  the  other  hand,  find  the  most  favorable  conditions  for  growth 
and  activity  upon  the  living  tissues  of  higher  forms  of  animal  life.  They 
include  most  of  the  so-called  pathogenic  or  disease-producing  bacteria. 

No  marked  separation  between  these  two  divisions  can  be  made, 
as  numerous  species  occupy  a  transition  point  between  the  two.  The 


84  INFECTION 

terms  are  merely  relative,  and  bacteria  ordinarily  saprophytic  may 
develop  parasitic  and  pathogenic  powers  when  the  resistance  of  the  host 
is  sufficiently  reduced  by  another  infection,  fatigue,  exposure,  or  other 
deleterious  influence.  In  other  words,  a  pathogenic  microorganism 
is  one  that  can  grow  in  the  living  tissues  because  the  immunologic 
defenses  of  the  host  are  not  sufficiently  strong  to  resist  it;  in  most  cases, 
however,  as  will  be  pointed  out  further  on,  a  higher  degree  of  immunity 
can  be  produced  artificially,  rendering  the  bacterium  in  question  rela- 
tively harmless  for  that  particular  animal.  Similarly,  under  certain 
circumstances,  the  resistance  of  the  body  or  of  a  part  of  it  may  be  broken 
down  to  such  an  extent  that  microorganisms  ordinarily  regarded  as 
saprophytes  may  gain  access  to  the  deeper  tissues,  flourish,  and  produce 
disease. 

Accordingly,  no  fundamental  distinction  between  pathogenic  and 
non-pathogenic  bacteria  can  be  made.  Any  apparent  differences  are 
due  not  only  to  various  degrees  of  pathogenicity  possessed  by  the  micro- 
organism, but  also  to  the  different  degrees  of  resistance  against  their 
attacks,  since  a  microparasite  that  is  highly  pathogenic  toward  one 
animal,  may  be  quite  harmless  to  another. 


CONTAGIOUS  AND  INFECTIOUS  DISEASES 

Just  as  all  pathogenic  bacteria  do  not  possess  the  same  habits  of 
growth,  so,  likewise,  they  vary  in  their  vitality  and  in  their  ability  to 
proliferate  under  various  conditions  when  removed  from  the  animal 
body.  Some  are  strictly  parasitic,  and  are  able  to  grow  only  at  body 
temperature,  or,  indeed,  only  in  the  human  body  itself;  when  removed 
from  these  conditions,  they  may  retain  their  vitality  for  a  short  period 
of  time,  but  are  unable  to  proliferate:  from  this  it  follows  that  com- 
munication of  these  bacteria  and  their  disease  must  be  direct  or  immedi- 
ate, i.  e.,  from  person  to  person,  or  almost  direct,  by  the  conveyance  of 
the  infecting  agent  in  the  form  offomites,  such  as  dust,  epidermal  scales, 
or  discharges,  or  as  the  result  of  bites  of  suctorial  insects.  This  form 
of  infection,  which  requires  such  direct  means  of  transmission,  and  of 
which  gonorrhea  is  an  example,  constitutes  what  are  known  as  conta- 
gious diseases. 

Other  microparasites  are  not  so  strictly  parasitic;  they  may  be  able 
to  preserve  their  pathogenic  powers  and  proliferate  outside  of  the  body 
at  ordinary  temperatures,  and  may  even  withstand  great  extremes  of 
heat  or  cold  and  various  nutritional  deficiencies;  they  may  exist  thus 
for  weeks,  and  carry  the  disease  to  a  second  individual  through  con- 


EXOGENOUS   AND   ENDOGENOUS   INFECTIONS  85 

taminated  material.     Infections  the  result  of  indirect  transmission  are 
known  as  infectious  diseases. 

There  are  no  hard-and-fast  rules  that  can  be  set  down  in  classifying 
bacterial  infections;  bacteria  that  are  commonly  transmitted  by  one 
means  may,  under  slightly  altered  conditions,  be  transmitted  by  another. 
The  usual  classification,  by  which  certain  diseases  are  classified  as  con- 
tagious and  others  as  infectious,  should  be  abolished,  and  all  should  be 
grouped  under  the  term  infectious,  there  being  a  definite  understanding  of 
those  cultural  characteristics  that  render  infection  more  likely  to  occur 
by  direct  and  immediate  contact,  and  those  that  may  occur  in  an  in- 
direct or  roundabout  manner.  It  may  therefore  be  stated  that  all  bac- 
terial diseases  are  infectious;  the  term  contagious  may  be  reserved  for 
those  spread  or  contracted  as  the  result  of  direct  contact. 


EXOGENOUS  AND  ENDOGENOUS  INFECTIONS 

Infection  may  occur  as  the  result  of  the  admission  of  microparasites 
to  the  tissues  from  sources  entirely  apart  from  the  individual  infected 
(exogenous  infection),  or  from  the  admission  of  some  of  those  micro- 
parasites  living  normally  and  harmlessly  on  the  skin  and  adjacent  mucous 
membranes,  and  which,  under  special  conditions,  have  assumed  patho- 
genic properties  (endogenous  infections). 

Exogenous  infections  are  the  more  usual  form,  and  result  from  con- 
tact with  infective  material  outside  the  body. 

1.  Microorganisms,  such  as  typhoid  and  cholera  bacilli,  which  can 
live  for  varying  periods  of  time  in  water  and  foods,  are  particularly 
likely  to  gain  entrance  through  the    gastro-intestinal   tract.     Micro- 
organisms may  be  present  in  milk  derived  directly  from  diseased  animals 
or  tissues,  .and  when  ingested,  may  produce  disease.     Thus,  for  example, 
the  germs  of  tuberculosis  may  be  conveyed  in  either  milk  or  flesh,  young 
children  being  particularly  exposed  to  this  method  of  infection. 

2.  The    atmosphere    may    be    laden   with   microorganisms,    which, 
whether  or  not  capable  of  proliferating  outside  of  the  body,  are  prone 
to  gain  entrance  through  the  respiratory  tract,  especially  through  the 
upper  air-passages,  the  pharynx  and  tonsils  being  often  the  seat  of  the 
infection. 

3.  Microorganisms  capable  of  existing  on  the  skin  may  gain  entrance 
to  the  deeper  tissues  as  the  result  of  wounds.     Under  these  conditions 
of  lowered  vitality  of  the  local  tissues    microorganisms   that  would 
otherwise  be  harmless  may  become  pathogenic  and  morbidly  affect  the 
host,  either  locally  or  generally.     As  the  skin  is  brought  so  freely  in 


86  INFECTION 

contact  with  external  objects,  various  microorganisms,  and  particularly 
the  pathogenic  cocci,  may  gain  entrance  to  the  dermis.  Wounds  may 
be  infected  by  the  teeth  and  secretions  of  animals,  or  by  various  weapons 
and  implements  contaminated  with  infective  material,  as,  e.  g.}  the  virus 
in  the  saliva  of  rabid  dogs,  or  the  spores  of  the  tetanus  bacillus  on  rusty 
nails. 

Contact  with  unclean  objects  of  various  kinds — eating  utensils, 
catheters,  syringes,  dental  instruments,  etc. — may  serve  to  transfer 
pathogenic  bacteria  from  one  person  to  another.  This  is  especially 
likely  to  occur  if  the  skin  or  mucous  membrane  is  abraded,  the  infecting 
parasites  thus  gaining  ready  access  to  the  deeper  tissues.  In  some  in- 
fections, however,  even  this  local  injury  is  unnecessary,  as  the  bacterium 
may  be  able  to  proliferate  and  produce  lesions  on  an  intact  surface,  as, 
for  instance,  the  diphtheria  bacillus  in  the  pharynx,  and  various  fungi, 
such  as  Achorion,  Trichophyton,  etc.,  on  the  scalp  and  skin  in  general. 

Microparasites  affecting  the  genital  organs  are  likely  to  be  conveyed 
directly  from  one  sex  to  the  other  in  conjugation,  or  to  the  child  during 
parturition. 

4.  Suctorial  insects  may  serve   as   the   medium   by  which   micro- 
organisms are  transmitted  from  person  to  person.    In  most  instances  the 
transmission  is  a  purely  mechanical  process,  as  witness  the  transmissions 
of  the  plague  bacillus  in  the  intestinal  contents  of  the  rat  flea;   in  the 
case  of  malaria,  on  the  other  hand,  the  interposition  of  the  mosquito  is 
essential  to  complete  the  life  cycle  of  the  protozoon. 

5.  Microorganisms  infecting  the  placenta  may  pass  to  the  fetus  by 
way  of  the  umbilical  vein. 

Endogenous  infections  arise  as  the  result  of  the  activity  of  micro- 
organisms having  their  normal  or  customary  habitat  in  the  body.  Such 
infections  do  not  represent  so  much  an  assumption  of  pathogenic  power 
on  the  part  of  the  microorganism,  as  they  do  a  disturbance  of  the  de- 
fensive mechanism  of  the  host,  whereby  the  normal  relations  are  dis- 
turbed, and  microorganisms  that  normally  are  harmless,  become  in- 
fective and  disease-producing.  While  the  disturbance  of  the  defensive 
mechanism  may  be  general,  it  is  far  more  likely  to  be  local ;  an  example 
is  that  of  appendicitis  the  result  of  Bacillus  coli  infection  following 
passive  congestion  due  to  fecal  impaction  of  the  colon. 

AVENUES  OF  INFECTION 

Local  infection  may  occur  in  any  portion  of  the  body,  and  any  part 
may  prove  the  point  of  entrance  of  bacteria  to  the  body  fluids,  the  result 


AVENUES   OF   INFECTION  87 

being  a  general  infection.  Owing,  however,  to  the  peculiar  pathogenic 
properties  of  different  bacteria  and  their  affinity  for  the  cells  of  certain 
tissues,  coupled  with  a  peculiar  tissue  susceptibility  for  certain  bacteria 
or  their  products,  we  find  that  many  diseases  have  regular  avenues  of  in- 
fection, and,  indeed,  in  a  few  instances  infection  of  the  human  body  may 
be  possible  only  through  a  particular  and  definite  route.  Infections 
of  the  gastro-intestinal,  respiratory,  and  genito-urinary  tracts  and 
various  sinuses  with  external  openings  must  be  considered  as  being 
potentially  surface  infections.  The  outer  layers  do  not  consist  merely 
of  the  skin  and  adjacent  mucous  membranes,  but  are  made  up  of  all 
layers  covering  surfaces  and  channels,  which,  however  indirectly,  com- 
municate with  the  exterior.  In  the  higher  animals  there  is  only  one 
direct  channel  of  communication  between  the  actual  interior  and  the 
exterior  of  the  body,  this  being  through  the  Fallopian  tube  of  the  female, 
which  normally  has  so  fine  a  lumen  and  is  so  well  protected  that  to  all 
intents  and  purposes  it  may  be  regarded  as  closed.  In  certain  inflam- 
matory conditions  of  the  genital  organs,  and  particularly  after  parturi- 
tion, the  Fallopian  tube  may  be  open,  and  afford  a  direct  route  for  the 
transmission  of  infection  from  the  external  parts  to  the  peritoneal 
cavity. 

Living  in  and  on  the  actual  and  potential  external  surfaces  are  count- 
less microorganisms,  which  are  for  the  most  part  harmless,  a  few  being, 
however,  actually  or  potentially  dangerous. 

1.  The  skin  and  adjacent  mucous  membranes,  particularly  in  those 
portions  where  warmth  and  moisture  abound,  are  well  adapted  to  bac- 
terial growth,  and  their  contact  with  surrounding  objects  causes  a  large 
variety  of  microorganisms  to  adhere  to  them. 

As  a  result,  the  bacteriology  of  the  skin  is  quite  complex,  since  it  may 
lodge  microorganisms  from  the  air,  from  water,  and  from  soil.  A  group 
of  cocci  and  diplococci,  particularly  the  Staphylococcus  epidermidis 
albus  of  Welch,  and  the  various  pseudodiphtheria  bacilli,  are  habitually 
present  upon  the  human  skin.  When  local  injury  occurs,  they  may 
produce  minor  suppurative  lesions,  and  may  be  concerned  in  the  pro- 
duction of  certain  skin  diseases,  such  as  eczema,  impetigo  contagiosa, 
the  pustules  of  variola,  etc, 

Other  microorganisms  may  find  temporary  lodgment  upon  the  skin, 
and  are  in  no  sense  regular  inhabitants.  For  example,  the  fingers  and 
hands  may  become  contaminated  with  colon,  typhoid,  and  tubercle 
bacilli,  pneumococci,  etc. 

The  skin  forms  a  very  important  barrier  against  the  entrance  of 


88  INFECTION 

bacteria  into  the  deeper  tissues.  The  greater  number  of  local  surgical 
infections  result  from  the  entrance  of  bacteria  into  lesions  of  the  skin, 
although  these  lesions  may  be  so  small  as  to  escape  notice. 

Certain  parasites  are  capable  of  producing  direct  action  on  the  skin 
without  previous  existing  injury,  and  especially  upon  the  mucous  mem- 
branes, where  moisture  and  higher  temperature  are  more  favorable  to 
bacterial  growth.  For  example,  a  few  of  the  higher  fungi,  such  as 
Microsporon,  Achorion,  and  Trichophyton,  seem  able  to  establish 
themselves  in  the  superficial  cells  and  invade  the  deeper  tissues  through 
the  hair-follicles;  staphylococci  may  reach  the  roots  of  hair-follicles 
and  sweat-glands  and  set  up  suppurative  conditions;  diphtheria  bacilli 
may  lodge  directly  on  the  intact  mucosa  of  the  upper  air-passages  and 
cause  local  necrosis  and  general  intoxication ;  cholera  bacilli  may  have  a 
similar  effect  upon  the  intestinal  mucosa;  the  Koch- Weeks'  bacillus 
and  the  gonococcus  may  produce  severe  inflammation  of  an  intact 
conjunctiva,  etc. 

2.  The  respiratory  organs  commonly  afford  admission  to  certain 
microorganisms.     The  nose  may  be  the  seat  of  local  infection  with 
Bacillus  influenzse,  Micrococcus  catarrhalis,   Bacillus  diphtherias,  and 
other  bacteria;   it  may  be  the  entrance  point  for  meningococci  and  the 
virus  of  anterior  poliomyelitis.     Similarly,  the  entrance  of  such  unknown 
infectious  agents  as  those  of  scarlet  fever,  measles,  and  smallpox  can 
best  be  accounted  for  by  assuming  that  they  were  inhaled  and  later 
entered  the  blood;  there  is  much  clinical  evidence  to  support  the  belief 
that  the  contagium  of  scarlet  fever  is  present  in  the  discharges  of  the 
upper  air-passages  of  persons  suffering  from  that  infection. 

Whether  or  not  tuberculosis  of  the  lungs  is  the  result  of  the  inhalation 
of  tubercle  bacilli  is  a  much-disputed  point,  but  it  cannot  be  denied  that 
this  theory  most  readily  accounts  for  the  far  greater  frequency  with 
which  tuberculosis  affects  the  lungs  than  it  does  other  organs  of  the  body. 

Pneumonia,  caused  by  the  pneumococcus  of  Weichselbaum,  probably 
results  from  the  direct  inhalation  of  one  of  the  various  types  of  pneumo- 
cocci,  and  bronchopneumonia  of  children  is  certainly  chiefly  inspiratory 
in  origin. 

3.  The  digestive  tract  may  be  the  portal  of  entrance  of  many  in- 
fections. The  mouth  usually  harbors  various  fungi  and  bacteria,which  may 
produce  local  infections,  and  either  directly  or  indirectly  cause  caries 
of  the  teeth.     The  -putrefactive  changes  they  may  produce  is  being 
generally  recognized  as  having  an  important  bearing  on  the  causation 
and  symptomatology  of  several  infections,  and  a  carious  tooth  has  been 


AVENUES   OF   INFECTION  89 

found  the  portal  of  entry  of  microorganisms  causing  a  general  infection. 
The  tonsils  are  well  known  to  be  the  breeding-  and  lodging-place  of 
various  microparasites  causing  many  general  infections,  such  as  acute 
rheumatic  fever,  tuberculosis,  and  possibly  typhoid  fever.  The  pharynx 
may  harbor  the  microorganisms  of  diphtheria,  pneumococcous  angina, 
etc. 

Normally,  except  for  the  presence  of  a  few  sarcinse,  the  stomach  is 
practically  sterile.  Under  special  conditions,  however,  typhoid,  dysen- 
tery, cholera,  tubercle,  and  other  infectious  bacteria  may  escape  the 
germicidal  effects  of  the  hydrochloric  acid,  and,  reaching  the  alkaline 
intestinal  contents,  which  are  rich  in  soluble  proteins  and  carbohydrates, 
are  rendered  capable  of  producing  their  respective  infections. 

Although  these  conditions  are  primarily  of  the  nature  of  local  in- 
fection, there  is  much  experimental  evidence  to  show  that  bacilli,  and 
particularly  tubercle  bacilli,  may  pass  through  a  practically  intact 
intestinal  wall  and  find  their  way  to  the  lymph-glands  or  to  the  blood- 
stream itself. 

Aside  from  these  direct  and  specific  infections,  various  other  micro- 
organisms, by  fermentative  action,  may  alter  the  intestinal  contents 
and  produce  toxic  products  capable  of  exciting  acute  and  severe  toxemias. 
Some  authorities  as,  e.  g.,  Metchnikoff,  regard  the  various  types  of  colon 
bacilli  as  producing  toxic  products  responsible  for  chronic  degenerative 
lesions  of  the  cardiovascular  and  other  organs.  The  digestive  tract  is 
therefore  regarded  by  some  pathologists  as  a  constant  menace  to  health, 
in  that  it  permits  bacteria  to  enter  the  lymphatic  and  blood-streams,  or 
to  produce  toxic  substances  detrimental  to  health  and  longevity.  Adami 
has  drawn  particular  attention  to  a  condition  which  he  terms  suhin- 
fection,  and  which  is  dependent  upon  the  constant  entrance  of  colon 
bacilli  into  the  blood,  whence  they  enter  the  liver,  where  their  final 
dissolution  takes  place,  appearing  as  fine,  dumb-bell-like  granules 
inclosed  in  the  cells. 

4.  The  genital  organs  are  the  seat  of  various  local  infections  that  may 
become  wide-spread  and  general.  Normally,  the  urethra  may  contain 
a  few  cocci  which  lodge  about  the  meatus;  the  acid  secretions  of  the 
vagina  are  generally  inimical  to  bacterial  growth,  and  the  uterus  and 
bladder  are  usually  sterile.  But  three  microorganisms — the  gonococcus, 
Treponema  pallidum,  and  the  bacillus  of  Ducrey,  here  find  favorable 
conditions  for  growth,  and  are  usually  transmitted  from  person  to  per- 
son by  means  of  sexual  congress.  The  local  gonococcal  lesion  may 
be  the  portal  of  entry  of  gonococci  into  the  blood-stream,  resulting  in 


90  INFECTION 

wide-spread  metastases  in  the  heart  valves  and  joints.  The  local  syphi- 
litic lesion  is  quickly  followed  by  general  infection.  Chancroids  alone 
remain  localized,  although  the  initial  lesion  frequently  spreads  quite 
rapidly  by  continuity  of  tissues.  In  rarer  instances  other  microorgan- 
isms, such  as  the  ordinary  pyogenic  cocci,  tubercle  bacillus,  and  diph- 
theria bacillus,  may  infect  these  organs  and  be  transmitted  by  sexual 
conjugation. 

5.  There  is  considerable  controversy  of  opinion  regarding  the  suscep- 
tibility of  the  placenta  and  the  filtering  properties  it  possesses  for  various 
infectious  agents.  A  study  of  this  subject  by  Nee' low1  would  indicate 
that  the  non-pathogenic  bacteria  do  not  pass  from  the  mother  through 
the  placenta  to  the  fetus.  Other  pathogenic  agents  may,  however,  pass 
through  quite  readily,  for  example,  pregnant  women  suffering  from 
smallpox  may  be  delivered  of  infants  showing  active  lesions  of  prenatal 
infection,  and  syphilitic  infection  of  the  fetus  is  a  well-known  condition. 
Most  controversy  centers  around  congenital  tuberculosis,  and  directly 
opposing  views  for  and  against  prenatal  infections  are  held  by  several 
authorities.  Baumgarten  is  of  the  opinion  that  many  children  are 
subject  to  antenatal  infection,  though  the  disease  infrequently  develops 
in  a  few  of  them. 

The  general  subject  of  antenatal  infection  and  pathology  is  a  field 
requiring  considerable  investigation  and  research. 


NORMAL  DEFENSES  AGAINST  BACTERIAL  INVASION 

When  the  large  area  of  the  body  that  is  subject  to  traumatic  injury 
and  accidental  infection  is  considered,  it  is  remarkable  that,  considering 
the  enormous  numbers  of  various  bacteria,  infection  does  not  occur  more 
frequently. 

Bacterial  invasion  of  the  tissues  is  of  frequent  occurrence,  but  in 
health  they  do  not  usually  cause  infection,  and  tend  to  be  destroyed  very 
soon  after  they  enter  the  tissues. 

It  may  be  well  to  discuss  at  this  point  the  factors  tending  to  prevent 
invasion,  and  leave  the  consideration  of  the  defensive  mechanism, 
whereby  the  body  destroys  bacteria  after  successful  invasion  and  thus 
prevents  infection,  for  the  chapter  on  Natural  Immunity. 

Of  the  factors  preventing  bacterial  invasion,  the  following  are  recog- 
nized: 

1.  The  structure  of  the  surface  layer  of  epithelium.  The  epidermal 
1  Centralb.  f.  Bakt.,  1902,  orig.,  xxxi,  691. 


MECHANISM    OF   BACTERIAL   INVASION  91 

cells  offer  a  mechanical  obstacle  to  invasion.  This  resistance  is  naturally 
more  complete  where  the  cells  are  thickened  and  most  compact.  In  the 
depths  of  glands  and  in  mucous  membranes,  where  numerous  glands  are 
present,  and  where  the  layers  are  thinner  and  moisture  exists,  the 
barrier  is  less  complete. 

2.  Surface   discharges  are  potent  factors  in  preventing  bacterial 
invasions  by — (1)  Washing  away  the  bacteria  mechanically;    (2)  by 
germicidal  activity  through  the  presence  of  various  chemical  agents, 
such  as  acids,  which  they  may  contain,  and  (3)    by  antiseptic  and 
even  bactericidal  substances  that  may  be  present  in  the  form  of  anti- 
bodies. 

The  saliva,  with  its  antiseptic  and  germicidal  properties,  is  potent 
in  preventing  infections  of  the  mouth  and  upper  air-passages;  when 
this  secretion  is  diminished,  as  during  the  course  of  high  fever,  bacterial 
activity  is  enhanced,  which  is  evidenced  by  the  development  of  fetid 
sordes  about  the  teeth  and  on  the  lips. 

The  acidity  of  the  gastric  juice  and  its  germicidal  powers  are  well 
known  and  appreciated;  similarly  the  urine,  the  milk,  and  to  a  slight 
extent,  the  bile,  have  been  demonstrated  by  "Adami  to  exert  a  distinct 
antiseptic  effect  upon  certain  bacteria,  such  as  the  Bacillus  coli. 

Surface  moisture  and  discharges  about  the  nose  and  throat  are  also 
potent  factors  in  mechanically  removing  bacteria  from  inspired  air,  and 
no  doubt  frequently  prevent  bacterial  invasion  of  the  lower  respiratory 
tract,  where  more  mischief  may  be  done. 

3.  The  cells  of  certain  excreting  glands  may  possess  bactericidal  and 
excretory  powers  of  value  in  preventing  bacterial  invasion  (Adami). 


MECHANISM  OF  BACTERIAL  INVASION 

We  will  now  consider  the  method  by  which  invasion,  the  first  step 
of  what  may  be  an  infection,  is  brought  about.  In  brief,  one  or  all  of 
the  normal  defenses  just  described  must  be  overcome;  in  some  instances 
the  microorganisms,  by  their  inherent  disease-producing  powers,  may 
accomplish  this  unaided;  in  other  instances  the  resistance  is  overcome 
by  a  general  lowering  of  the  vitality  of  the  body  defenses. 

1.  Traumatic  solution  of  the  surface  layers  of  epithelial  cells  is  a 
very  important  factor  in  the  production  of  infection,  as  the  invading 
microparasites  are  thus  given  easier  access  to  the  deeper  and  less  resistant 
tissues.  The  pathologist  or  surgeon  may,  in  the  course  of  his  work, 
contaminate  his  hands  with  secretions  containing  virulent  microorgan- 


92  INFECTION 

isms,  and  may  yet  escape  infection,  unless  a  small  break  in  the  surface 
epithelium,  in  the  form  of  a  scratch  or  a  needle-prick,  is  present. 

2.  As   has  been  previously  stated,   certain  bacteria,   notably  the 
diphtheria  bacillus,  by  concentrating  at  one  point,  may  lower  the  vitality 
and  cause  necrosis  of  superficial  cells  of  the  mucosa  lining  the  upper  air- 
passages,  and  in  this  manner  induce  a  local  break  in  the  continuity  of  the 
epithelial  covering.     Staphylococci  may  exert  a  similar  action  in  the 
depths  of  sweat  and  sebaceous  glands,  and,  indeed,  certain  fungi,  such 
as   the    Trichophyton,    Microsporon,   and   Achorion,   may  attack  the 
intact  skin.     While,  therefore,  solution  of  the  surface  coverings  is  a  very 
important  source  of  many  infections,  it  is  not  essential  for  the  produc- 
tion of  all. 

3.  Alterations  of  the  surface  discharges,  either  in  quantity  or  in 
quality,  may  permit  bacteria  to  proliferate  freely  and  produce  sufficient 
toxic  matter  to  affect  the  surface  cells,  lower  their  vitality,  and  destroy 
them,  with  the  result  that  they  may  gain  entrance  to  the  deeper  tissues. 
When  the  secretions  are  diminished  or  altered,  as,  for  example,  the  saliva 
during  a  fever,  unless  the  mouth  is  carefully  and  frequently  cleansed, 
it    becomes    putrescent    with    bacterial    growth.     Similarly    catarrhal 
gastritis,  or  any  other  factor  tending  to  lower  acidity  of  the  gastric  juice, 
favors  infection  by  this  route. 

4.  Not  infrequently  bacteria  may  gain  access  to  the  deeper  tissues 
or  to  an  internal  organ,  and  infection  may  occur  without  any  recognizable 
solution  of  continuity  of  the  surface  epithelium.     In  these  hidden  or 
"cryptogenic  infections"  the  entrance  point  of  the  parasites  may  be 
healed  over,  or  the  infecting  microorganisms  may  have  been  carried  to 
the  circulating  body  fluids  by  the  wandering  cells. 

Not  infrequently,  in  cases  of  tuberculosis  of  the  cervical  and  mesen- 
teric  glands  in  children,  there  may  be  no  signs  whatever  of  local  irrita- 
tion in  the  fauces  or  in  the  intestine  to  explain  the  source  of  infection. 
The  tonsils  are  now  strongly  suspected,  and  indeed  known  to  be,  the 
source  of  entry  of  bacteria  causing  several  acute  and  chronic  infections. 

The  leukocytes,  in  their  phagocytic  activities,  no  doubt,  play  an 
important  role  in  the  production  of  cryptogenic  infections,  especially 
when  an  excessive  number  of  pathogenic  bacteria  have  congregated  at 
one  point,  and  congestion,  increased  leukocytic  infiltration,  and  a 
lowered  vitality  of  the  tissues  have  occurred  prior  to  the  invasions  of 
microorganisms.  Wandering  cells  are  commonly  found  on  mucous 
membranes,  gathering  up  various  bacterial  and  cellular  debris.  They 
may  carry  a  virulent  microorganism  into  the  deeper  tissues,  and,  al- 


MECHANISM    OF   BACTERIAL   INVASION  93 

though  this  may  not  produce  an  infection,  a  large  number  of  bacteria  so 
transported  may  be  able  to  resist  destruction  and  prove  capable  of 
causing  infection. 

5.  Aside  from  the  question  of  local  conditions  in  the  process  of  in- 
fection, other  factors  may  exert  an  influence.  The  temperature  of  the 
host  may  be  unsuitable  for  the  growth  of  a  certain  parasite,  even  though 
it  has  gained  entrance  to  the  deeper  tissues;  a  particular  route  for  the 
introduction  of  the  infecting  agents  may  be  necessary,  as  in  typhoid 
fever  and  cholera,  which  are  probably  always  intestinal  infections,  and, 
finally,  even  after  the  infecting  agent  has  reached  the  deeper  tissues, 
extension  is  prevented  by  a  local  inflammatory  reaction.  In  many  such 
instances  the  question  of  natural  immunity  is  brought  into  intimate 
relation  with  the  subject  of  infection. 

After  invasion  has  occurred,  some  bacteria  can  best  sustain  them- 
selves against  the  defenses  of  the  host  at  the  local  point  of  entry.  Such 
microorganisms  may,  however,  possess  unusual  vitality,  and  indirectly, 
through  the  lymphatics,  find  their  way  to  the  blood-stream,  producing 
a  bacteremia.  This  is  a  morbid  condition  characterized  by  the  presence 
of  microorganisms  in  the  circulating  blood. 

Some  microorganisms  may  gain  entrance  to  the  general  circulation 
more  readily  than  others,  and  their  mode  and  route  of  entry  vary  in  the 
different  infections.  It  is  essential  that  they  possess  an  unusual  degree 
of  invasive  power,  and  be  capable  of  protecting  themselves  against  the 
manifold  defensive  factors  contained  in  the  blood.  Kruse  believes  that 
in  local  infections  the  high  pressure  of  an  inflammatory  exudate  may 
force  bacteria  into  the  adjacent  vessels;  that  they  may  sometimes  be 
carried  into  the  deeper  tissues,  and  even  into  the  blood-stream,  by 
leukocytes  is  not  to  be  denied. 

When  bacteria  have  entered  the  circulation,  they  may  act  as  emboli 
in  the  finer  capillaries,  or,  being  unable  to  remain  in  the  circulation,  may 
collect  in  the  capillaries  of  less  resistant  tissues,  proliferating  and  pro- 
ducing local  metastatic  lesions,  usually  purulent  in  character.  The 
condition  thus  produced  is  known  as  pyemia. 

Saprophytic  bacteria  or  pathogenic  bacteria  of  feeble  invasive  powers 
may  be  able  to  grow  in  diseased  tissues,  such  as  gangrenous  areas,  and 
may  assist  in  effecting  morbid  changes,  producing  toxic  products  of 
decomposition,  which  when  absorbed  into  the  body,  give  rise  to  a  series 
of  toxic  phenomena,  such  as  fever,  rapid  pulse,  malaise,  etc.  This  con- 
dition is  known  as  sapremia,  a  term  that  has  also  been  applied  to  the 
decomposition  of  relatively  sterile  organic  material  and  absorption  of 


94  INFECTION 

the  toxic  products,  as  when  portions  of  placenta  or  fetal  membranes 
are  retained  in  the  uterus  after  childbirth. 

The  term  toxemia  is  employed  rather  loosely  to  mean  the  presence 
of  any  toxic  material.  Its  use  should  be  limited  to  the  condition  re- 
sulting from  the  absorption  of  the  poisonous  substances  produced  by  the 
non-invasive  bacteria  themselves,  as  in  diphtheria  and  tetanus.  Septi- 
cemia  is  the  term  applied  to  the  presence  in  the  body-fluids  of  toxic  products 
generated  by  the  pyogenic  microorganisms. 

FOCAL  INFECTION 

In  recent  years  considerable  attention  has  been  given  the  subject 
of  focal  infection,  that  is,  the  primary  focus  of  such  systemic  diseases  as 
acute  rheumatic  fever,  endocarditis,  chorea,  and  chronic  rheumatoid 
arthritis  may  be  located  in  the  head  and  usually  in  the  form  of  alveolar 
abscesses  and  acute  and  chronic  tonsillitis  and  sinusitis.  Foci  of  infection 
may  also  be  located  in  the  genito-urinary  and  intestinal  tracts  and  care- 
ful bacteriologic  examinations  of  sputa,  urine,  and  urethral  discharges 
and  exudates  of  synovial  cavities, 'of  excised  lymph-nodes  proximal  to 
the  infected  regions  and  of  bits  of  infected  muscles  and  tendons,  have 
often  yielded  results  of  diagnostic  value  and  indicated  the  etiologic 
relation  of  the  focus  of  infection  to  existing  systemic  infection.  The 
subject  is  one  of  considerable  importance  and  deserving  of  continued 
clinical  and  bacteriologic  investigation.  An  excellent  review  of  this 
subject  based  mainly  upon  the  investigations  of  the  Chicago  School  of 
Clinicians  and  Bacteriologists  has  been  recently  made  by  Billings.1 

MECHANISM  OF  INFECTION 

Since  bacterial  invasion  is  of  frequent  occurrence,  the  question 
naturally  arises,  Why  are  not  infections,  both  local  and  general,  more 
frequent?  Thus  abrasions  of  the  surface  epithelium  are  not  uncommon 
in  the  presence  of  active  microorganisms;  tubercle  bacilli  may  be  in- 
spired, and  typhoid  bacilli  may  be  swallowed,  the  altered  local  con- 
ditions affording  opportunity  for  producing  infection,  and  yet  the  host 
may  escape. 

Bacterial  invasion,  therefore,  does  not  necessarily  mean  infection,  and 
it  may  be  stated  that  infection  can  only  take  place  when — 

(1)  The  microorganisms  are  sufficiently  virulent. 

(2)  When  they  invade  the  body  by  appropriate  avenues  and  reach 
susceptible  tissues. 

1  Focal  Infection;  The  Lane  Medical  Lectures,  Appleton  and  Co.,  1916. 


VIRULENCE  95 

(3)  When  they  are  present  in  sufficient  numbers. 

(4)  When  the  host  is  generally  susceptible  to  their  action. 

(5)  When  the  microorganisms  are  able  to  resist  the  defensive  forces 
of  the  host  through  special  agencies  aside  from  their  offensive  forces. 

Not  all  these  factors  must  necessarily  be  present  before  infection  may 
occur.  A  microorganism  may  be  particularly  virulent,  so  that  numbers 
are  relatively  unimportant;  a  host  or  a  portion  of  the  host  may  be  so 
susceptible  or  vulnerable  to  infection  that  a  microorganism  of  low 
virulence,  which,  under  normal  conditions,  would  be  totally  unable  to 
produce  infection,  may  now  prove  pathogenic. 

VIRULENCE 

Virulence  refers  to  the  disease-producing  power  of  a  microorganism, 
and  is  dependent  upon  two  variable  factors:  (1)  Toxicity,  and  (2)  aggres- 
siveness, or  the  invasive  power  of  the  bacteria.  In  most  infections  usually 
both  factors  are  operative. 

Toxicity  is  the  term  applied  to  the  kind  and  amount  of  poison  or  toxin 
produced.  This  poison  may  be  readily  soluble,  or  exogenous,  diffusing 
into  the  surrounding  tissues  and  being  readily  absorbable;  or  it  may  be 
endogenous,  and  contained  chiefly  within  the  microorganisms,  and  be 
liberated  only  upon  the  dissolution  of  the  bacterial  cell. 

Aggressiveness  is  a  term  applied  to  the  invasive  powers  of  a  micro- 
organism to  enter,  live,  and  multiply  in  the  body-fluids,  or,  in  other  words, 
to  the  aggressive  or  progressive  forces  of  the  microorganism  in  its  new 
environment. 

Toxicity  is  generally  confused  with  aggressiveness,  a  highly  toxic 
microorganism  being  regarded  as  an  aggressive  one.  For  example,  the 
bacillus  of  tetanus  is  highly  toxic  because  of  the  production  of  a  potent 
soluble  poison  which  gives  rise  to  the  symptoms  of  tetanus,  although 
it  is  only  slightly  aggressive,  being  almost  unable  to  multiply  in  the 
tissues.  The  anthrax  bacillus,  on  the  other  hand,  is  highly  aggressive, 
owing  to  the  fact  that  it  usually  multiplies  to  such  an  extent  that  it  can 
be  found  in  each  drop  of  blood  and  in  every  organ  of  an  infected  animal; 
nevertheless  it  is  but  slightly  toxic,  the  animals  frequently  showing  few 
or  no  symptoms  until  shortly  before  death.  The  toxicity  of  a  micro- 
organism should,  therefore,  be  regarded  separate  from  its  aggressive- 
ness, although  in  many  infections  both  factors  are  so  intimately  con- 
cerned that  the  term  virulence  may  be  used  to  express  the  degree  of 
pathogenicity  or  the  total  disease-producing  power. 

The  virulence  of  a  microorganism  is  more  or  less  specific,  i.  e.,  the 


96  INFECTION 

toxin  produced  by  one  species  is  different  from  that  produced  by  another 
in  the  kind  of  disease  produced  and  the  species  of  animal  infected. 
Some  toxins  are  active  for  certain  animals  only  and  not  for  others. 
Microorganisms  of  one  group  may  possess  general  and  common  patho- 
genic properties  differing  only  in  degree;  those  of  different  morphologic 
and  cultural  characters  may  possess  totally  different  powers. 

The  virulence  of  a  given  species  is  subject  to  great  variation.  A  few 
bacteria  almost  constantly  retain  their  virulence,  even  when  kept  for 
years  under  artificial  conditions;  as  an  example  may  be  mentioned  the 
diphtheria  bacillus;  others  quickly  lose  their  virulence  as  soon  as  they 
are  grown  artificially,  as,  e.  g.,  the  influenza  bacillus;  in  others — and 
probably  the  larger  class — the  virulence  may  be  raised  or  lowered  ac- 
cording to  the  experimental  manipulations  to  which  they  may  be  sub- 
jected. Variations  may  also  be  observed  among  members  of  the  same 
group  of  microorganisms,  and  even  among  individual  microorganisms 
of  the  same  strain. 

Decrease  of  virulence  of  a  microorganism  may  be  brought  about 
artificially  by  repeated  growth  in  or  upon  culture-media,  especially 
when  transfers  to  fresh  media  are  made  at  prolonged  intervals.  This 
decrease  probably  depends  upon  an  actual  decrease  in  virulence,  and 
particularly  upon  the  selection,  in  artificial  growth,  of  the  less  virulent 
or  vegetative  forms  which  grow  actively  and  soon  exceed  in  number 
their  more  pathogenic  fellows.  Each  time  the  culture  is  transplanted 
more  of  the  vegetative  and  fewer  of  the  pathogenic  microorganisms 
are  carried  over,  until  finally  the  pathogenic  bacteria  are  entirely  elim- 
inated, or  their  virulence  totally  destroyed,  and  the  entire  culture  is 
composed  only  of  vegetative  or  harmless  forms  of  bacteria. 

Various  other  agencies  lead  to  artificial  lessening  of  virulence,  such 
as  exposure,  for  short  periods  of  time,  to  a  temperature  just  under  the 
thermal  death-point;  exposure  to  sunlight;  exposure  to  small  quantities 
of  antiseptic  or  germicidal  substances;  the  action  of  desiccation;  sub- 
jection to  increased  atmospheric  pressure,  etc.,  these  methods  being  com- 
monly employed  in  the  preparations  of  vaccines  to  be  used  for  purposes 
of  active  immunization. 

The  passage  of  a  microorganism  or  virus  through  animals  usually 
increases  its  virulence,  but  may  modify  or  attenuate  it,  as  in  the  case  of 
the  passage  of  smallpox  virus  through  the  calf,  when  it  loses  forever  its 
power  of  producing  smallpox. 

Increase  in  virulence  can  best  be  secured  by  passing  the  microorgan- 
ism through  animals.  It  is  practically  impossible,  by  any  means,  to 


VIRULENCE  97 

make  a  known  non-virulent  microorganism  virulent,  although  it  is 
comparatively  easy  to  increase  the  virulence  of  a  culture  that  has  be- 
come well-nigh  non-virulent  on  account  of  prolonged  artificial  cultiva- 
tion. This  fact  is  worthy  of  emphasis,  and  is  well  illustrated  by  the 
large  amount  of  work  that  has  been  done  in  fruitless  attempts  to  render 
non-virulent,  diphtheria-like  bacilli  virulent  by  passage  through  various 
animals  or  growth  on  special  culture-media. 

In  cases  where  the  virulence  is  slight  or  absent,  experimental  manip- 
ulations of  the  culture  are  directed  toward  gradual  immunization  of  the 
microorganisms  to  the  defensive  mechanism  of  the  body  of  the  animal 
for  which  the  organism  is  to  be  made  virulent.  This  is  well  explained 
according  to  the  hypothesis  of  Welch,  and  will  be  referred  to  again  in 
the  latter  part  of  this  chapter.  A  number  of  methods  are  made  use  of 
for  this  purpose: 

(a)  Passage   through   animals,   which   enables   the   microorganisms 
gradually  to  immunize  themselves  or  adopt  certain  morphologic  and 
biologic  changes  enabling  them  best  to  resist  the  defensive  forces  of  the 
host.     Since  these  defensive  forces  vary  with  different  animals,  and 
indeed  with  the  various  organs  of  the  same  animals,  it  is  usual  to  find 
that  virulence  raised  by  animal  passage  affects  only  the  animal  or  the 
particular  organ  of  a  certain  animal,  and  not  all  animals  in  general. 
Thus,  in  general,  the  passage  of  bacteria  through  rabbits  increases  their 
virulence  for  rabbits  and  not  for  mice,  dogs,  pigeons,  etc.;    passage 
through  mice  may  increase  their  virulence  for  mice,  but  not  for  rabbits, 
guinea-pigs,  etc. 

(b)  The  use  of  collodion  sacs  for  increasing  virulence  has  been  ad- 
vocated,  especially  by  French  investigators.     When  microorganisms 
are  inclosed  in  a  collodion  capsule  of  the  proper  thickness  and  placed 
within  the  abdominal  cavity  of  a  suitable  animal,  the  slightly  modified 
body-juices  are  able  to  transfuse  through  the  sac,  impeding  the  develop- 
ment of  such  microorganisms  as  are  unable  to  immunize  themselves  or 
withstand  the  injurious  influences.     In  this  manner  a  race  of  virulent 
bacteria  are  artificially  selected  which  can  endure  the  defensive  agencies 
of  those  juices  with  which  they  have  come  into  contact. 

(c)  The  addition  of  animal  fluids  to  the  culture-medium  may  enable 
the  bacteriologist  to  maintain  or  even  to  increase  the  virulence  of  a 
microorganism  according  to  the  principles  of  artificial  selection.     The 
fluid,  either  a  serum  or  whole  blood,  is  secured  in  a  sterile  manner  and 
added  to  the  medium  in  a  raw  or  unheated  condition.     In  this  manner 
the  microorganisms  are  exposed  to  some  of  the  defensive  agencies  con- 


98  INFECTION 

tained  in  the  juices  under  these  conditions,  and  this  tends  to  destroy 
the  less  resistant  bacteria,  encourage  the  more  resistant,  and  at  least 
maintain,  for  a  longer  or  a  shorter  time,  the  virulence  of  a  culture 
freshly  isolated  from  a  lesion  or  cultivated  by  animal  passage. 


THE  AVENUE  OF  INFECTION  AND  TISSUE  SUSCEPTIBILITY 

Successful  infection  of  the  body  by  certain  bacteria  can  be  accom- 
plished only  when  invasion  takes  place  through  appropriate  avenues. 
Thus  typhoid,  cholera,  and  dysentery  infection  seems  to  take  place 
through  the  gastro-intestinal  tract,  and  doubtfully  by  inhalation,  and 
not  at  all  through  the  skin  or  urogenital  system;  gonococci  usually 
enter  the  body  through  the  genital  organs  or  the  eye,  and  not  through 
the  respiratory  apparatus  or  through  the  skin.  The  route  of  infection 
is  less  important  with  microorganisms  characterized  by  great  aggres- 
siveness and  producing  general,  rather  than  local,  infections.  For 
example,  in  most  animals  anthrax  is  a  general  bacteremia,  regardless 
of  the  route  of  invasion;  plague  rapidly  becomes  a  bacteremia,  whether 
the  bacilli  are  inhaled,  rubbed  into  the  skin,  or  reach  the  lymphatics 
through  superficial  abrasions;  similarly,  local  staphylococcus  and 
streptococcus  infection  may  become  general,  regardless  of  the  route  of 
invasion  or  the  location  of  the  local  lesion. 

The  avenue  of  invasion  is  also  of  importance  in  determining  the  form, 
nature,  and  virulence  of  an  infection.  Thus  virulent  pneumococci 
lodging  in  the  pharynx  may  produce  a  pseudomembranous  angina;  in 
the  eye,  a  severe  conjunctivitis;  and  in  the  lungs,  a  pneumonia.  When 
tubercle  bacilli  gain  admission  through  the  skin,  they  may  produce  lupus, 
or  a  low-grade  inflammatory  disease  rarely  terminating  fatally.  When 
inhaled,  they  may  produce  tuberculosis  of  the  lungs;  in  the  throat  they 
may  reach  the  tonsils  and  later  the  local  lymphatic  glands,  etc.  When 
swallowed,  they  may  produce  ulceration  of  the  intestines,  or  pass  through 
the  intestinal  walls  and  involve  the  mesenteric  glands,  and  later  the 
lungs  or  other  organs. 

Just  as  general  susceptibility  of  the  host  renders  infection  more 
likely  to  occur,  so  local  susceptibility  may  be  induced  by  injury  and 
fundamental  disorders.  These  changes  may  not  only  furnish  pabulum 
for  the  invading  bacteria,  but  more  especially  reduce  the  local  resistance 
of  the  body  defenses. 

Even  more  important,  however,  is  the  predisposition  of  some  patho- 
genic microorganisms  to  attack  certain  tissues  or  organs,  and  the  fact 


THE    AVENUE    OF    INFECTION    AND    TISSUE    SUSCEPTIBILITY  99 

that  these  tissues  are  particularly  weak  in  defensive  power,  so  that  the 
bacteria  naturally  lodge  where  conditions  are  most  favorable  for  their 
growth. 

Selective  Tissue  Affinity. — While  the  primary  focus  of  infection  is 
determined  largely  by  the  route  of  invasion,  the  selective  affinity  of  micro- 
organisms or  their  toxins  for  certain  tissues  and  the  inherent  tissue  sus- 
ceptibility to  the  toxins  are  best  in  evidence  in  the  location  of  secondary 
foci  or  localization  of  the  infection  in  general  bacterernias.  Thus  the  seat 
of  the  principal  local  lesions  in  pneumonia  is  the  lungs,  and  in  typhoid 
fever  the  lymphoid  tissues,  especially  that  of  the  spleen,  and  Fever's 
patches  in  the  intestine.  It  is  true  that  mechanical  factors  may  aid  in 
this  selection,  as,  e.  g.,  the  occlusion  by  emboli  of  microorganisms  caught 
in  the  capillaries  of  organs;  but,  in  general,  we  must  conclude  that  either 
— (1)  Microorganisms  tend  to  be  destroyed  in  every  tissue  or  organ 
except  those  that  are  poor  in  defensive  forces  and  are  susceptible,  or  (2) 
that  microorganisms  or  their  products  circulate  passively  through  a 
tissue  and  do  not  lodge  because  they  possess  no  affinity  for  these  cells. 
In  many  infections  both  processes  are  probably  operative,  and  at  least  we 
are  led  to  the  very  important  conclusion,  laid  down  by  Adami,  that  "in 
infections  the  body  is  never  involved  as  a  whole.  Coincidentally  with 
the  growth  of  the  specific  germs  in  individual  organs  there  tends  to  be  a 
reaction  to,  and  destruction  of,  the  same  in  other  parts." 

Nickols  and  Hough1  and  Reasoner2  have  isolated  strains  of  Treponema 
pallidum  from  the  nervous  tissues  that  appeared  to  have  selective  affin- 
ities for  the  cornea,  choroid,  and  retina  of  the  eyes  of  rabbits;  Noguchi3 
has  noticed  that  various  types  produced  lesions  in  rabbits  of  certain 
distinct  characters  and  with  considerable  constancy.  Further  and  simi- 
lar studies  may  show  that  various  strains  of  Treponema. pallidum  may 
possess  selective  tissue  affinities  and  thereby  explain  the  early  develop- 
ment of  lesions  in  the  central  nervous,  cardiovascular,  and  cutaneous 
systems  of  different  persons.  According  to  the  investigations  of  Rose- 
now4  various  bacteria,  and  particularly  streptococci,  exhibit  extreme 
degrees  of  tissue  affinity  and  produce  various  constant  and  distinct 
lesions  in  rabbits  after  inoculation  by  various  routes. 

The  numeric  relationship  of  bacteria  to  infection  is  very  important, 
and  the  number  alone  may  determine  whether  or  not  it  shall  occur. 

1  Jour.  Amer.  Med.  Assoc.,  1913,  50,  108. 

2  Jour.  Amer.  Med.  Assoc.,  1916,  56,  1917. 

3  Jour.  Lab.  and  Clin.  Med.,  1917,  2,  472. 

4  Jour.  Infect.  Dis.,  1915,  16,  240;  ibid.,  1915,  16,  367;  ibid.,  1915,  17,  219;  ibid., 
1915,  17,  403;  Jour.  Amer.  Med.  Assoc.,  1914,  63,  1835;  ibid.,  1915,  64,  1968. 


100  INFECTION 

Usually  the  normal  defensive  factors  of  the  body  are  sufficient  to  over- 
whelm one  or  a  few  bacteria  unless  they  are  especially  virulent.  When 
an  inter  current  or  chronic  disease,  malnutrition,  or  injury  renders  the 
host  more  susceptible  than  normal,  fewer  bacteria  than  would  other- 
wise be  required  may  successfully  infect  the  body.  Also  with  true 
parasites,  or  those  with  well-marked  aggressiveness,  such  as  the  anthrax 
bacillus,  a  few  may  be  sufficient,  if  they  reach  the  circulating  fluids,  to 
produce  infection.  Thus  Webb,  Williams,  and  Barbor1  have  found  that 
one  anthrax  bacillus  was  sufficient  to  infect  a  white  mouse,  and  as  few 
as  20  tubercle  bacilli  were  sufficient  in  one  instance  to  infect  a  guinea-pig. 

Likewise  Kolmer,  Schamberg,  and  Raiziss2  have  found  in  experi- 
mental trypanosomiasis  that  the  infection  of  white  rats  by  intraperi- 
toneal  injection  with  varying  numbers  of  trypanosomes,  counted  after 
the  method  of  Kolmer,3  greatly  modifies  the  time  of  appearance  of  try- 
panosomes in  the  peripheral  blood  and  the  duration  of  life,  and  has  an 
important  bearing  upon  studies  in  the  chemotherapy  of  experimental 
trypanosomiasis. 

Park  has  directed  attention  to  the  fact  that  when  bacteria  are  trans- 
planted from  culture  to  culture,  under  supposedly  favorable  conditions, 
many  of  them  die;  it  is  highly  probable  that  when  they  are  transplanted 
to  an  environment  that  is  likely  to  be  unfavorable,  as  are  the  body 
tissues  with  various  defensive  mechanisms,  many  more  must  die.  This 
is  an  important  point  to  bear  in  mind  in  attempting  to  correlate  experi- 
mental results  with  the  natural  cause  of  an  infectious  disease.  In  the 
laboratory  we  reproduce  disease  experiment  ally,  by  the  immediate  in- 
jection of  millions  of  bacteria,  whereas  in  nature  there  is  rarely  any  such 
immediate  overwhelming  of  the  tissues.  For  example,  pneumonia  may 
be  produced  experimentally  in  dogs  by  the  injection  of  a  large  number  of 
virulent  pneumococci  directly  and  at  once  into  the  bronchi,  yielding  a 
positive  result  with  a  microorganism  which,  under  natural  conditions 
and  in  smaller  numbers,  would  be  relatively  innocuous  for  the  animal 
under  observation. 

GENERAL  SUSCEPTIBILITY  IN  RELATION  TO  INFECTION 
Under  normal  conditions  the  body-cells  of  a  host  will  invariably 
offer  some  resistance  to  invasion  and  infection  by  pathogenic  micro- 
organisms.    When,  however,  any  condition  that  depresses  or  diminishes 
general  physiologic  activity  and  vitality  exists,  the  host  may  be  unable 

1  Transactions  Sixth  International  Congress  on  Tuberculosis,  1908,  p.  194. 

2  Jour.  Infect.  Dis.,  1917,  20,  10;  ibid.,  1917,  20,  35. 

»  Jour.  Infect.  Dis.,  1915,  16,  311;  ibid.,  1915,  17,  79. 


GENERAL   SUSCEPTIBILITY   IN    RELATION   TO    INFECTION  101 

to  master  these  defensive  forces,  and  accordingly  becomes  predisposed 
or  more  susceptible  to  infection. 

Predisposition  may  be  inherited  or  acquired. 

Inherited  predisposition  may  be — -(a)  Specific,  or  species  suscep- 
tibility, as,  e.  g.,  dogs  to  distemper;  cattle  to  contagious  pleuropneu- 
monia;  hogs  to  hog  cholera;  man  to  gonorrhea;  chancroids,  acute 
exanthemata,  typhoid  fever,  etc.  (6)  Racial,  as  Eskimos  to  measles 
and  syphilis,  ordinary  sheep  to  anthrax,  whereas  Algerian  sheep  are 
immune,  etc.  Racial  susceptibility  is  frequently  but  a  lack  of  acquired 
immunity;  for  instance,  measles,  syphilis,  gonorrhea,  and  other  diseases 
brought  by  settlers  to  foreign  peoples  among  whom  these  diseases  were 
previously  unknown,  find  them  peculiarly  susceptible  and  the  diseases 
unusually  virulent,  (c)  Familial,  i.  e.,  members  of  a  family  may, 
through  generations,  be  unusually  susceptible  to  scarlet  fever,  tuber- 
culosis, rheumatism,  rheumatoid  arthritis,  metabolic  disturbances,  etc. 
(d)  Individual  predisposition,  which  depends  principally  upon  sex,  age, 
and  peculiar  tissue  susceptibility.  Thus  infants  are  especially  prone  to 
contract  certain  infections  on  account  of  the  immature  development  of 
the  body-cells,  and  this  susceptibility  to  infection  is  further  influenced 
by  acquired  factors,  chiefly  malnutrition.  On  the  other  hand,  very 
young  children  enjoy  an  immunity  to  several  infections,  such  as  typhoid 
fever,  scarlet  fever,  and  even  diphtheria,  probably  due,  as  Abbott  has 
suggested,  to  the  fact  that  pathogenic  substances  that  may  set  up  molec- 
ular and  destructive  disturbances  in  the  poorly  developed  cell  have  but 
little  effect  upon  the  more  inert  protoplasm  of  the  immature  cell,  and 
that  if  certain  bacteria  gain  admission  to  the  tissues,  the  cells  may 
destroy  them,  their  toxins  not  combining  with  the  molecular  side-chains, 
and,  as  a  consequence,  not  injuring  or  interfering  with  the  cell  functions. 

Acquired  susceptibility  bears  a  more  important  relation  to  infection, 
and  may  be  due  to  various  factors,  most  of  which  lead  to  a  state  of  re- 
duced vitality,  normal  physiologic  processes  being  impaired  to  a  greater 
or  less  degree. 

(a)  Overwork  or  overstrain  leads  to  general  or  local  predisposition 
to  disease.  Those  engaged  in  hard  labor,  mental  or  physical,  which 
involves  late  hours  and  inadequate  periods  of  rest  and  recreation,  fre- 
quently associated  with  inadequate  nutrition  and  foul  air,  are  likely  to 
succumb  to  tuberculosis,  typhoid  fever,  pneumonia,  etc. 

The  influence  of  overstrain  on  acute  infections  has  been  shown  ex- 
perimentally by  Charrin  and  Roger,1  who  found  that  white  rats  natur- 
ally immune  to  anthrax  became  quite  susceptible  after  being  compelled 
1  Compt.  rend.  Soc.  de  Biol.  de  Paris,  January  24,  1890. 


102 


INFECTION 


to  turn  a  revolving  wheel  until  exhausted  before  they  were  inoculated; 
similarly  of  four  guinea-pigs  who  were  placed  in  a  cage  so  constructed 
that  they  were  forced  to  keep  moving  for  one  or  two  days  three  died  in 
from  two  to  nine  days  after  the  experiment.  Smears  and  cultures  made 
from  the  livers,  spleens,  and  blood  gave  positive  results. 

(6)  Previous  infection  with  the  same  or  another  infectious  disease 
may  predispose  the  individual  to  renewed  infection.  Thus  some  in- 
fections, such  as  erysipelas,  furunculosis,  acute  rheumatism,  pneumonia, 
and  influenza,  not  only  fail  to  leave  the  body-cells  immune,  but  actually 
predispose  to  second  attacks.  Whether  the  microorganisms  of  these 
diseases  are  not  all  destroyed,  but  are  retained  in  the  system  and  become 
active  when  the  general  vitality  is  lowered,  or  whether  a  new  infection 
occurs,  is  not  definitely  known,  and  probably  either  may  occur. 

One  attack  of  an  infectious  disease  may  weaken  the  tissues  and 
render  them  susceptible  to  an  infection  of  a  different  nature.  Thus 
the  acute  exanthemata  may  follow  one  another,  and  tuberculosis  may 
supervene  upon  any  of  them. 

(c)  Malnutrition  exerts  some  effect  on  the  resistance  to  infection. 
Thus  the  terrible  epidemics  of  plague,  cholera,  typhus  fever,  and  typhoid 
fever  which  have  followed  in  the  wake  of  famines  in  Europe  and  Asia 
during  the  past  centuries  are  examples  of  the  influence  of  malnutrition 
as  a  factor  in  predisposing  to  disease.     The  tendency  of  marasrriatic 
infants  to  develop  enterocolitis,  thrush,  bronchopneumonia,  and  other 
infections,  and  of  scorbutics  to  local  infections  of  the  mouth,  illustrates 
the  influence  of  insufficient  food  in  decreasing  the  resistance  to  disease. 
Here  may  also  be  included  local  malnutrition,  such  as  loss  of  nerve  or 
blood  supply,  predisposing  to  local  infection,  especially  with  pyogenic 
microorganisms. 

(d)  Diet  produces  some  variation  in  the  resisting  powers  to  infection. 
For  example,  the  ordinary  wild  rat  is  not  susceptible  to  anthrax  unless 
it  is  fed  for  a  week  or  more  on  coarse  dry  food,  when  it  become  sus- 
ceptible.    Here,  of  course,  malnutrition  may  come  in  intimate  relation- 
ship with  diet,  as  an  inefficient  diet  may  greatly  lower  the  general 
resistance.     The  influence  of  diet  is  particularly  noticeable  from  the 
fact  that  the  diseases  of  carnivorous  animals  are  not  the  same  as  those 
that  affect  herbivorous  animals,  and  that  each  class  is  frequently  im- 
mune to  some  of  the  diseases  that  attack  the  other. 

(e)  Intoxications  of  various  kinds  predispose  to  infections.     Thus  it 
is  a  common  clinical  observation  that  excessive  indulgence  in  alcoholic 
beverages  predisposes  to  infections,  notably  pneumonia.     Abbott1  has 

.  Exper.  Med.,  1896,  1,  No.  3. 


DEFENSIVE    MECHANISM    IN    RELATION   TO    INFECTION  103 

demonstrated  experimentally  that  the  daily  administration,  to  rabbits, 
of  5  to  10  c.c.  of  alcohol  introduced  into  the  stomach  by  a  tube,  renders 
these  animals  more  susceptible  to  infection  with  Streptococcus  pyogenes 
and  Bacillus  coli.  Wagner,  Leo,  and  Platania  have  also  found  animals 
that  under  the  influence  of  chloral,  phloridzin,  alcohol,  and  curare  are 
more  susceptible  to  infection. 

(/)  Exposure  to  cold  and  wet  frequently  lowers  the  resistance  of 
man  and  other  warm-blooded  animals  to  infection.  The  influence  of 
these  factors,  well  illustrated  in  the  etiology  of  " colds"  and  pneumonia, 
is  not  without  experimental  foundation.  Thus  Pasteur  found  that 
fowls,  which  are  naturally  immune  to  anthrax,  are  readily  infected  if 
they  are  inoculated  after  their  body  temperature  has  been  reduced  by 
a  cold  bath.  Conversely,  Gibier1  has  shown  that  frogs,  which  are  also 
naturally  immune  to  anthrax,  are  readily  infected  if  their  temperature 
is  previously  elevated  and  maintained  at  37°  C. 

(</)  Trauma  and  morbid  conditions  in  general  may  predispose  to  in- 
fection. Thus  injuries  reduce  the  local  resistance  and  facilitate  local 
infections  that  vary  with  the  severity  and  extent  of  the  trauma.  The 
increased  susceptibility  of  injured  joints  and  pneumonic  lungs  to  tuber- 
culosis; the  frequent  and  oftentimes  extensive  streptococcus  infection 
accompanying  scarlet  fever  and  smallpox;  the  increased  susceptibility 
of  diabetics  to  furunculosis  and  local  gangrenous  lesions  of  the  skin — 
all  show  the  increased  susceptibility  of  individuals  already  injured  or 
diseased  to  infection. 


THE  DEFENSIVE  MECHANISM  OF  THE  MICROORGANISM  IN  RELATION 

TO  INFECTION 

After  bacterial  invasion  has  occurred,  the  question  of  whether  or 
not  the  microorganism  can  overcome  the  defensive  forces  of  the  host  and 
prove  pathogenic  may  depend  to  some  extent  upon  the  peculiar  defensive 
factors  of  the  invading  bacteria  against  the  offensive  mechanism  of  the 
host,  aside  from  their  toxins  or  other  distinctly  offensive  forces. 

Morphologic  and  Physiologic  Changes  of  the  Microorganisms. — For 
example,  capsule  formation  or  thickening  of  the  ectoplasm  of  certain 
bacteria  is  evidence  of  their  increased  powers  of  resistance  against  the 
opposing  forces  of  the  host.  The  capsule  may  be  quickly  lost  when  the 
microorganism  is  cultivated  on  artificial  media-,  and  its  virulence  be  cor- 
respondingly lowered,  but  by  repeated  animal  inoculations  a  race  of 

1  Compt.  rend.  Acad.  de  Sci.  de  Paris,  1882,  xcix,  1605. 


104  INFECTION 

capsulated  organisms  with  increased  virulence  is  produced,  explaining 
in  a  way  the  mechanism  of  animal  passage  in  raising  the  virulence  of  a 
given  organism.  This,  however,  is  not  invariable,  and,  indeed,  may  act 
in  a  contrary  manner,  as  the  passage  of  smallpox  virus  through  heifers 
attenuates  and  modifies  instead  of  increasing  its  virulence. 

Aggressins. — The  microorganism  may  actively  secrete  a  material 
that  overwhelms  the  defensive  forces  of  the  host.  This  phase  of  the 
subject  has  been  studied  exclusively  by  Bail,  who  sought  to  prove  that 
the  question  of  pathogenicity  of  a  microorganism  is  dependent  upon  its 
ability  to  secrete  substances  that  are  able  to  paralyze  the  protective 
forces  of  the  host,  especially  the  leukocytes.  These  substances  are 
called  "  aggressins, "  and  they  were  distinguished  by  the  fact  that  they 
were  formed  by  living  bacteria  and  only  in  the  living  body.  In  support 
of  this  theory  Bail  was  able  to  show  that  substances  are  present  in  the 
exudates  of  fatal  infections,  which,  when  injected  in  small  quantities 
into  another  animal  with  sublethal  doses  of  the  microorganism,  would 
cause  a  rapidly  fatal  infection.  Later  Wassermann  and  Citron  showed 
that  " artificial  aggressins"  could  be  prepared  by  autolyzing  bacteria 
in  water  or  serum.  While  the  subject  of  aggressins  is  still  unsettled, 
there  is  strong  evidence  to  show  that  they  are  the  endotoxins  liberated 
by  the  breaking-up  of  the  microorganism. 

The  well-known  statement  of  Metchnikoff,  that  a  particular  virulent 
microorganism  is  not  so  readily  taken  up  by  leukocytes  as  is  an  avirulent 
strain,  may  be  explained  by  the  fact  that  the  microorganism,  in  its 
virulent  parasitic  state,  secretes  substances  that  repel  the  phagocytes, 
neutralize  the  opsonins,  or  form  actual  leukocytic  toxins.  This  action 
may  be  due  to  liberated  endotoxins,  or,  as  Bail  claims,  to  specific  secre- 
tory substances  of  the  bacterium — the  aggressins — specifically  formed 
and  liberated  by  the  microorganism  for  protection  against  the  host. 

Hypothesis  of  Welch. — Not  entirely  foreign  to  this  subject  is  the 
very  interesting  hypothesis  of  Welch.  A  bacterium  may  not  only  pro- 
duce substances  directly  inimical  to  the  defensive  forces  of  the  host,  but 
it  may  actually  immunize  itself  against  these  defensive  powers.  "  Looked 
at  from  the  point  of  view  of  the  bacterium,  as  well  as  from  that  of  the 
animal  host,  according  to  the  hypothesis  advanced,  the  struggle  between 
the  bacteria  and  the  body-cells  in  infections  may  be  concerned  as  an 
immunizing  contest  in  which  each  participant  is  stimulated  by  its  op- 
ponent to  the  production  of  cytotoxins  hostile  to  each  other,  and  thereby 
endeavors  to  make  itself  immune  against  its  antagonist." 

It  is  well  known  that,  when  freshly  isolated  from  a  patient  having 


DEFENSIVE    MECHANISM    IN    RELATION   TO    INFECTION  105 

typhoid  fever,  the  typhoid  bacillus  resists  agglutination,  whereas  it 
becomes  easily  agglutinable  after  a  period  of  artificial  cultivation.  It 
may  be  assumed  that,  when  active,  the  bacillus  as  an  infecting  agent 
gradually  became  more  resistant  against  the  agglutinating  properties 
of  the  patient's  serum,  and  that,  when  grown  on  artificial  media,  it  loses 
this  resistance  by  being  removed  from  the  stimulating  influence  of  the 
infected  body. 

This  hypothesis,  however,  would  go  a  step  further  in  assuming  the 
possibility  of  the  receptors  of  the  invading  bacteria  anchoring  certain 
constituents  of  our  body-fluids,  and  being  stimulated  to  the  production 
of  various  cytotoxins,  which  attack  the  leukocytes,  erythrocytes,  nerve- 
cells,  liver,  kidney,  etc.  In  other  words,  each  bacterium  may  be  con- 
ceived as  being  composed  of  a  central  atom  group  with  numerous  side- 
chains,  just  as  Ehrlich  conceived  the  hypothetic  structure  of  body-cells, 
and  that  these  side-chains,  primarily  present  for  the  purpose  of  anchor- 
ing food  material,  may  likewise  anchor  various  pathogenic  animal  sub- 
stances, with  the  production  of  substances  acting  as  antibodies  to  the 
opposing  forces  of  the  host.  Welch  assumed  that  these  bodies  were  of 
the  nature  of  amboceptors,  which  may  become  complemented  by  bac- 
terial complement  or  by  endocomplements  of  the  tissue-cells;  this  is  of 
secondary  importance,  and  there  is  no  reason  why  they  may  not  be  of 
different  structure,  and  similar  to  all  three  orders  of  antibodies  produced 
by  body-cells  according  to  Ehrlich's  side-chain  theory  of  immunity. 

This  hypothesis  may  possibly  explain  certain  instances  of  so-called 
species  and  organ  virulence,  whereby  the  virulence  of  an  organism  arti- 
ficially increased  by  repeated  passage  through  animals  of  the  same  spe- 
cies, does  not  manifest  this  increased  virulence  for  animals  of  different 
species.  If,  for  example,  the  virulence  of  the  chicken  cholera  bacillus 
is  increased  by  repeated  passage  through  the  chicken,  the  increase  of 
virulence  affects  this  animal,  but  does  not  affect  the  guinea-pig.  Certain 
organs  may  likewise  be  subject  to  a  similar  selective  virulence,  if  the 
increase  in  virulence  has  been  induced  by  the  specific  intervention  of 
those  organs,  and  this  selective  virulence  shows  itself,  irrespective  of 
the  manner  in  which  the  infection  was  produced. 

That  virulence  of  this  order  is  playing  an  important  role  in  the  pro- 
cesses of  infection  is  a  theory  supported  by  the  discovery  that  different 
strains  of  the  same  species  of  bacteria  are  found  to  produce  characteristic 
lesions,  and  while  this  affinity  for  a  certain  organ  may  be  natural  and 
inherent,  there  can  be  no  doubt  that  it  may  also  be  experimentally 
induced  and  acquired.  For  example,  according  to  Rosenow,  a  certain 


106  INFECTION 

strain  of  streptococcus  will  produce  arthritis;    another,  endocarditis; 
another,  gastric  ulcer,  etc. 

This  remarkable  species  and  organ  specificity  may  be  due  to  the  fact 
that  the  bacteria  of  a  particular  culture  have  been  immunized  against 
defensive  forces  of  a  particular  animal  host  or  a  certain  organ  of  the  host, 
so  that,  when  introduced,  they  thrive  as  a  result  of  their  special  and 
acquired  offensive  forces.  On  the  other  hand,  the  specificity  may  be 
due  to  the  fact  that  the  bacteria  have  been  accustomed  to  a  certain 
nutriment  furnished  by  a  particular  species  or  organ,  and  that  they 
cannot  thrive  unless  they  receive  this  special  nutriment,  and,  as  a  result, 
the  species  or  organ  fulfilling  this  requirement  will  become  the  special 
seat  of  infection  (Simon) . 

MIXED  INFECTION 

Several  different  microorganisms  may  produce  infection  at  the  same 
time,  or  one  may  follow  the  other  or  others  and  produce  secondary 
infection.  The  combined  effects,  upon  the  tissues  of  the  host,  of  the 
products  and  action  of  two  or  more  varieties  of  pathogenic  bacteria, 
and  also  of  the  influence  of  these  different  forms  on  each  other,  are  of 
great  importance  in  the  production  of  disease.  The  metabolic  products 
of  one  bacteria  may  neutralize  or  accelerate  the  action  of  an  associated 
species,  or  combine  to  form  a  new  substance  entirely  different  from  its 
antecedents. 

Thus  pyogenic  cocci  affect  anthrax  bacilli  in  an  injurious  manner; 
on  the  other  hand,  aerobic  bacteria  accelerate  or  make  possible  the 
growth  of  anaerobes  by  absorbing  uncombined  oxygen.  Tetanus 
bacilli  will  not  grow  outside  of  the  body  in  the  presence  of  oxygen  unless 
aerobic  bacteria  are  associated  with  them;  not  infrequently  tetanus 
bacilli  and  their  spores  would  not  develop  in  wounds  were  it  not  for  the 
presence  of  the  aerobic  bacteria  introduced  with  them;  this  factor  is  of 
much  importance,  especially  in  tetanus  produced  by  cowpox  vaccine, 
where,  through  careless  treatment  of  the  lesion,  both  tetanus  bacilli  and 
pyogenic  cocci  are  admitted  to  the  wround. 

Again,  it  may  be  found  that  one  microorganism  increases  the  viru- 
lence of  another;  thus  the  scarlet-fever  virus  is  favorable  to  the  develop- 
ment of  streptococci. 

Generally  all  infections  of  mucous  membranes  are  mixed  infections. 
Numerous  bacteria  are  present  upon  the  mucosa  of  the  air-passages 
and  gastro-intestinal  tract;  these  are  usually  harmless,  unless  the  resist- 
ance of  the  host  is  lowered  in  some  manner,  in  which  case  not  only  one 


SUMMARY  107 

but  several  varieties  of  these  bacteria  invade  the  tissues  and  cause 
infection.  When  one  pathogenic  microorganism,  such  as  the  typhoid 
bacillus,  has  caused  the  primary  infection,  because  of  the  local  and  gen- 
eral conditions  of  lowered  vitality  of  the  tissues,  these  otherwise  sa- 
prophytic  bacilli  tend  to  intensify  the  infection.  Blood  infections,  on 
the  other  hand,  are  usually  due  to  one  form  of  bacteria,  and  even  when 
two  or  more  varieties  are  introduced,  only  one,  as  a  rule,  is  capable  of 
surviving  and  developing.  The  products  of  certain  bacteria,  on  the 
other  hand,  may  immunize  the  host  against  infection  with  other  bacteria, 
for,  as  shown  by  Pasteur,  attenuated  chicken-cholera  cultures  may 
produce  immunity  against  anthrax.  In  the  intestine  harmless  varieties 
of  bacteria  may  be  made  to  crowd  out  more  dangerous  ones;  this  is 
exemplified  by  the  ingestion  of  soured  milk  which  contains  lactic-acid 
bacteria,  as  advocated  by  Metchnikoff. 

SUMMARY 

From  what  has  been  said  it  is  clear  that  infection  differs  from  mere 
surface  contamination,  and  cannot  be  said  to  occur  until  the  invading 
bacteria  have  reached  the  deeper  tissues,  or  a  point  where  they  may  grow 
and  multiply.  The  surface  epithelium  and  various  secretions  offer  the 
most  potent  local  obstacles  to  infection,  but  even  when  these  barriers 
are  broken  down,  the  invaders  may  not  survive  the  onslaughts  of  various 
protective  agencies  of  the  host.  In  order  to  withstand  and  overcome 
these  attacks,  the  bacterium  may  undergo  certain  morphologic  and 
physiologic  changes,  and  actively  secrete  a  substance  that  is  inimical 
to  the  defensive  forces  of  the  host,  or  immunize  itself  against  these  forces. 
Thus  a  certain  species  of  bacteria  may  become  selectively  fortified  or 
immunized  against  a  certain  host  or  organ  of  that  host,  and  show  a 
specific  affinity  for  producing  infection  of  a  certain  animal  or  a  particular 
organ.  When  the  bacterium  has  overcome  the  defensive  forces  of  a  host, 
it  may,  by  the  formation  and  action  of  exogenous  and  endogenous  toxins, 
bacterial  proteins,  mechanical  blocking  of  vessels,  or  formation  of  pto- 
mains,  produce  disease.  These  various  factors  will  be  considered  in 
greater  detail  in  the  following  chapter. 


CHAPTER  VII 
INFECTION  (Continued) 

PRODUCTION  OF  DISEASE 

WHEN  pathogenic  microorganisms  have  reached  the  deeper  tissues  and 
multiplied,  infection  has  occurred,  but,  as  previously  stated,  tissue 
changes  of  sufficient  extent  to  produce  definite  lesions  and  symptoms 
of  disease  may  or  may  not  result,  depending  upon  whether  or  not  the 
defensive  forces  of  the  host  are  able  to  overcome  the  invaders  or  are 
overcome  by  them.  If  the  latter  has  occurred,  and  the  invading  bac- 
terium is  firmly  established  in  its  host,  the  question  of  how  the  bacterium 
and  its  products  cause  disease,  that  is,  the  mechanism  of  the  production 
of  an  infectious  disease,  arises  for  consideration. 

The  subject  is,  indeed,  quite  complex.  Although  the  etiologic 
relationship  of  a  large  number  of  pathogenic  bacteria  to  definite  patho- 
logic changes  and  conditions  has  been  proved  by  the  regularity  with 
which  they  are  found  in  the  diseased  tissues,  and  in  many  instances  has 
been  corroborated  by  animal  experimentation,  yet  the  ways  and  means 
by  which  these  bacteria  produce  disease  are  quite  varied,  and  are  seldom 
dependent  upon  one  product  of  bacterial  activity.  For  example, 
diphtheria  and  tetanus  are  apparently  simple  infections,  being  caused 
by  soluble  toxins  secreted  by  the  respective  bacilli.  There  are  many 
factors  concerning  the  action  of  these  toxins,  however,  which  are  not  as 
yet  understood.  Again,  the  lesions  of  staphylococcus  and  other  pyo- 
genic  infections  are  probably  due  to  the  activities  of  soluble  toxins, 
endotoxins,  and  the  protein  of  the  bacterial  bodies.  All  three  of  these 
factors  are  probably  concerned  in  the  production  of  typhoid  fever  and 
cholera,  whereas  the  symptoms  of  sleeping  sickness  are  due  in  part  to 
blocking  of  a  small  but  physiologically  important  vessel  in  the  brain  by 
trypanosomes,  with  the  absorption,  at  the  same  time,  of  toxins  and  dis- 
integration products.  To  these  may  be  added  the  effects  of  other 
biologic  activities  of  the  bacteria  in  living  or  dead  tissues,  such  as  the 
production  of  gas  from  carbohydrates,  proteins,  etc.,  in  Bacillus  aero- 
genes  capsulatus  infection.  Each  infection,  therefore,  must  be  regarded 
as  largely  a  law  unto  itself,  so  that  all  that  will  be  included  within  the 
scope  of  this  book  will  be  the  mention  and  illustration  of  what  are 

108 


TOXINS  109 

generally  accepted  as  the  ways  and  means  by  which  bacteria  and  proto- 
zoa produce  disease,  omitting  a  description  of  each  disease  in  detail. 

In  the  great  majority  of  instances  disease  is  produced  as  the  result 
of  chemical  substances  generated  by  the  metabolic  processes  of  bacteria. 
Animal  parasites  and  certain  bacteria,  such  as  that  of  anthrax,  may  do 
harm  mechanically  by  forming  capillary  emboli;  but,  as  stated,  bac- 
teria, as  a  rule,  produce  their  effects  chiefly  through  chemical  means. 
Accordingly,  bacteria  may  give  rise  to  infection  and  disease  through  the 
following  agencies : 

1.  Soluble  or  extracellular  toxins,  which  are  poisons  generated  by 
bacterial  cells  and  discharged  into  their  surrounding  media. 

2.  Intracellular  toxins  or  endotoxins,  which  are  specific  poisonous 
products  of  bacterial  activity,  and  are  contained  within  the  cells. 

3.  Aggressins,  or  substances  secreted  by  bacteria,  that  neutralize 
opsonins  and  prevent  phagocytosis. 

4.  Bacterial  proteins,  which  are  poisonous  protein  constituents  of  the 
bacterial  cells  and  are  responsible  for  certain  general  and  non-specific 
lesions. 

5.  Ptomains,  which  are  the  secondary  products  of  decomposition  of 
the  media  upon  which  the  bacteria  are  growing;  these  may  be  absorbed 
and  produce  symptoms  of  intoxication. 

6.  Mechanical  action  of  bacteria,   whereby  certain  symptoms  or 
lesions  may  be  due  to  the  blocking  of  small  but  physiologically  important 
vessels  with  emboli  of  bacteria,  in  addition  to  the  effects  of  mechanical 
irritation. 

TOXINS 

Nomenclature. — Of  all  the  various  means  whereby  bacteria  produce 
disease,  none  possesses  so  much  importance  as  the  poisonous  substances, 
known  as  toxins,  elaborated  by  the  metabolic  activities  of  the  micro- 
organisms. A  few  classes  of  bacteria  secrete  this  poisonous  principle 
directly  into  the  tissues  or  artificial  culture-media  in  which  they  are 
growing,  and  hence  are  known  as  soluble,  exogenous,  extracellular,  or 
true  toxins.  Other  bacteria  retain  most  of  their  toxins  within  the 
bacterial  cell,  and  for  this  reason  are  called  endotoxins,  or  intracellular 
toxins;  these  are  liberated  upon  the  disintegration  of  the  bacteria  by 
various  mechanical,  physical,  or  chemical  means. 

By  common  consent  the  term  " toxin"  is  applied  to  the  soluble  or 
true  toxins,  such  as  those  of  diphtheria  and  tetanus,  and  hence  the  term, 
when  used  without  further  qualifications,  may  be  considered  to  refer  to 
toxins  of  this  class. 


110  INFECTION 

Aside  from  bacterial  toxins,  characteristic  poisons  are  also  produced 
by  certain  of  the  higher  plants  (phytotoxins)  and  animals  (zootoxins), 
and  although  few  are  of  medical  interest,  their  study  has  thrown  con- 
siderable light  on  the  phenomena  of  toxin-antitoxin  immunity. 

Extracellular  Bacterial  Toxins. — Definition. — Bacterial  toxins  may  be 
defined  as  poisonous  products  produced  by  bacteria  in  both  living  tissues  and 
artificial  culture-media.  The  symptoms  resulting  from  their  activity  appear 
after  a  certain  period  of  incubation,  and  all  are  capable  of  stimulating 
the  production  of  specific  antitoxins.  They  represent  the  chief  poison- 
ous product  of  bacteria,  and  are  mainly  responsible  for  the  symptoms 
of  infection  caused  by  the  specific  bacteria  that  have  produced  them. 

The  true  toxins  causing  infection  in  man  are  chiefly: 

1.  Diphtheria  toxin. 

2.  Tetanus  toxin. 

3.  Botulism  toxin  (a  form  of  meat  poisoning). 

4.  Dysentery  toxin  (Kruse-Shiga). 

5.  Staphylolysin,  streptolysin,  and  other  bacterial  toxins. 
General  Properties  of  Soluble  Toxins.— Many  of  the  true  toxins  are 

extremely  labile,  and  susceptible  to  the  action  of  heat,  light,  age,  etc.; 
consequently  an  absolutely  pure  toxin  is  practically  unknown.  Oxygen, 
even  as  it  occurs  in  the  air,  is  harmful;  all  oxidizing  agents,  including 
the  oxidizing  enzymes,  quickly  destroy  them,  and  Pitini1  has  ascribed 
the  harmful  effects  of  toxins  to  their  power  of  reducing  the  oxidizing 
capacity  of  the  tissues.  Some  substances  seem  to  attack  only  the 
toxophore  portion  of  the  toxin  molecule,  e.  g.,  iodin  and  carbon  disulphid 
(Ehrlich).  In  the  preparation  of  antitoxin,  the  first  doses  of  toxin  are 
frequently  modified  by  adding  a  chemical  of  this  nature.  According 
to  Gerhartz 2  z-rays  tend  to  weaken  the  toxins. 

Because  of  their  great  lability,  the  toxins  do  not  lend  themselves  to 
accurate  chemical  analysis.  Our  knowledge  of  them  has  been  gained 
largely  through  a  study  of  the  lesions  and  symptoms  produced  by  in- 
jecting the  toxins  into  susceptible  animals. 

They  are,  so  far  as  known,  uncrystallizable  and  thereby  differ  from 
ptomains;  they  are  soluble  in  water  and  dialyzable  through  thin  but  not 
thick  membranes.  They  are  precipitated  along  with  peptones  by 
alcohol,  and  also  by  ammonium  sulphate. 

The  toxins  are  all  poisonous,  but  in  order  to  exert  their  toxic  effect 
they  must  enter  into  chemical  combination  with  cells;  hence  there  is  a 
necessary  period  of  incubation  before  symptoms  of  their  activity  appear. 
1  Biochem.  Zeit.,  1910,  25,  257.  2  Berl.  klin.  Woch.,  1909,  46,  1800. 


TOXINS  111 

Most  bacterial  toxins  are  not  absorbed  from  the  intestine  (botulinus 
toxin  excepted),  and  when  introduced  into  the  gastro-intestinal  tract, 
they  are  usually  unable  to  produce  symptoms  and  are  quickly  destroyed. 

An  essential  property  of  a  toxin  lies  in  the  fact  that  we  can  immunize 
a  subject  against  it,  and  are  able  to  demonstrate  the  presence  of  antitoxin 
within  the  serum  of  the  immunized  animal. 

Chemical  Properties  of  Soluble  Toxins. — As  has  just  been  stated, 
the  exact  chemical  nature  of  toxins  is  unknown.  This  is  due  principally 
to  the  fact  that  pure  toxins  of  bacteria  are  rarely  obtainable,  except  in 
conjunction  with  their  associated  products,  such  as  lysins,  pigments,  acids, 
etc.,  as  well  as  to  the  great  lability  of  the  toxins.  A  summary  of  the 
results  of  researches  into  the  chemical  nature  of  toxins  would  indicate 
that  they  are  tcJxalbumins,  albumoses  or  allied  to  the  albumoses.  Certain 
investigators  have  reported  that  very  active  toxins  obtained  by  purifi- 
cation processes  did  not  give  the  protein  reactions,  yet  toxins  are  digested 
by  proteolytic  ferments,  and,  like  proteins,  are  precipitated  by  nucleic 
acid  (Kossel).  According  to  Field  and  Teague,1  the  toxins  act  like  electro- 
positive colloids,  but  diffuse  faster  than  do  proteins.  Our  present  knowl- 
edge of  the  chemistry  of  the  true  toxins  has  been  expressed  thus  by  Oppen- 
heimer:  "We  must  be  contented  to  assume  that  they  are  large  molecular 
complexes,  probably  related  to  the  proteins,  corresponding  to  them  in 
certain  properties,  but  standing  even  nearer  to  the  equally  mysterious 
enzymes  with  whose  properties  they  show  the  most  extended  analogies 
both  in  their  reactions  and  in  their  activities." 

Precipitation  of  the  Extracellular  Toxins. — After  the  toxin  has  been 
secured  by  filtration,  crystals  of  ammonium  sulphate  are  added  in  large 
excess  over  the  saturation  point,  and  the  whole  kept  at  37°  C.  for  eight- 
een hours.  The  toxin  is  precipitated  and  rises  to  the  surface  along  with 
the  albumoses  and  peptones.  This  is  skimmed  off  and  quickly  dried 
with  an  electric  fan  and  cold  air.  The  residue  is  ground  into  a  fine  pow- 
der and  stored  in  vacuum  tubes  kept  at  a  low  temperature  and  in  a  dark 
place.  During  this  process  there  may  be  considerable  deterioration 
and  especially  with  tetanus  toxin.  Banzhaf  has  obtained  highly  potent 
and  dried  diphtheria  toxin  by  slightly  acidulating  the  toxin  broth  and 
adding  absolute  ethyl  alcohol  up  to  65  per  cent.;  after  an  hour  or  two 
the  slight  precipitate  is  filtered  off,  quickly  dried,  and  kept  in  ampules. 

Structure  of  Toxins. — According  to  Ehrlich,  the  toxin  molecule 
consists  of  a  main  central  atom  or  radical,  with  a  large  number  of  organic 
side-chains  grouped,  as  in  other  organic  compounds,  about  this  main 

1  Jour.  Exper.  Med.,  1907,  9.  86. 


112  INFECTION 

radical.  Each  of  the  side*  or  lateral  arms  is  composed  of  two  portions — 
one,  the  haptophore  group,  which  has  a  chemical  affinity  for  certain 
chemical  constituents  of  the  tissues  of  susceptible  animals,  and  the  other, 
the  injury-producing  portion,  called  the  toxophore  group.  (See  Fig.  40.) 
An  animal  is  susceptible  to  a  toxin  only  when  its  cells  contain  substances 
that  possess  a  chemical  affinity  for  the  haptophore  group  of  the  toxin, 
and  also  substances  susceptible  to  the  toxic  action  of  the  toxophore  group. 

The  toxophore  group  is  far  more  unstable  and  susceptible  to  dele- 
terious influences  than  is  the  haptophore  portion.  When  the  molecule 
has  lost  the  toxophore  radical,  it  is  known  as  a  toxoid,  which  is  still 
capable  of  uniting  with  the  side  arms  of  cells  but  is  devoid  of  toxic  action. 

Nature  of  Toxins. — It  has  been  abundantly  demonstrated  that 
toxins  are  colloids,  and  in  many  respects  bear  a  close  resemblance  to 
enzymes.  (See  p.  254.)  The  toxins  are  synthetic  products  of  bacterial 
activity.  They  are  of  absolutely  specific  nature,  and  in  this  manner 
differ  from  ptomains,  which  are  cleavage  products  from  the  medium 
upon  which  the  bacteria  have  been  grown.  Furthermore,  ptomains  of 
similar  properties  may  be  produced  by  several  different  kinds  of  bacteria, 
and  accordingly  are  non-specific  in  nature.  Toxins,  like  ferments,  can 
give  rise  to  antibodies,  whereas  ptomains  cannot  produce  them. 

The  extracellular  or  soluble  toxins  differ  from  the  intracellular  toxins 
in  that  they  are  more  easily  diffused  throughout  the  animal  juices,  and 
that  their  diffusion  occurs  independently  of  the  invasiveness  of  the 
bacteria,  so  that  comparatively  few  microorganisms  growing  at  some 
unimportant  focus,  and  causing  but  slight  local  lesions,  may  be  able  to 
give  rise  to  profound  general  intoxication.  This  is  well  illustrated  in 
diphtheria,  where  the  local  lesion  in  the  throat  may  be  quite  small, 
and  in  tetanus,  where  it  may  indeed  be  undiscoverable — yet  either, 
through  the  action  of  their  toxins  on  special  tissues,  may  cause  profound 
intoxication  and  death. 

Selective  Action  of  Toxins. — Extensive  studies  of  the  toxins  of 
diphtheria  and  tetanus  and  of  cobra  venom  have  shown  that  they  are 
quite  complex,  and  are  usually  composed  of  two  or  more  distinct  and 
separate  toxins  possessing  different  pathogenic  properties,  although  one 
of  these  may  predominate  in  producing  symptoms. 

All  infections  with  the  group  of  true  toxin-producing  bacteria  mani- 
fest certain  non-specific  symptoms  of  general  intoxication,  namely, 
fever,  headache,  malaise,  prostration,  etc.;  but  the  typical  symptoms 
of  these  diseases  are  due  to  the  remarkable  selective  action  of  the  toxins 
upon  certain  cells  or  organs,  dependent  upon  the  ability,  chemical, 
physical,  or  both,  of  the  toxin  to  combine  with  these  specific  cells.  For 


SPECIAL   PROPERTIES    OF   THE    PRINCIPAL   TOXINS  113 

example,  tetanus  toxin  contains  tetanospasmin,  that  has  a  special 
affinity  for  nervous  tissue;  and  tetanolysin,  a  poison  that  has  a 
selective  affinity  for  erythrocytes  and  is  hemotoxic.  Ehrlich  has  shown 
that  these  are  really  different  toxins,  and  not  one  toxin  with  a  two-fold 
function,  even  the  antitoxins  of  the  two  being  different.  Similarly,  the 
general  symptoms  and  necroses  of  diphtheria  are  attributed  to  the  main 
toxin  of  the  bacillus,  and  the  nerve  lesions  and  paralyses  to  a  secondary 
but  distinct  secretory  product  known  as  toxon.  This  latter  view  of 
Ehrlich's,  however,  is  much  disputed,  many  investigators  believing  that 
toxon  represents  a  degenerated  or  modified  form  of  the  one  toxin. 

The  special  affinities  of  toxins  for  certain  tissues  have  analogies 
among  the  poisons  of  higher  plant  life,  as,  for  example,  strychnin  has  a 
similar  selective  affinity  and  is  said  to  be  specific  in  its  action  upon  the 
motor  cells. 

The  venom  of  various  serpents,  especially  that  of  the  cobra,  has 
specific  action:  the  erythrocytes  of  various  animals  are  readily  attacked 
by  it,  and  the  cells  of  the  respiratory  center  are  apparently  profoundly 
affected. 

Aside  from  the  special  effects  of  the  toxins  upon  certain  cells  and 
tissues,  it  must  be  remembered  that  toxins  may  involve  the  body-cells 
in  general,  and  particularly  those  of  the  parenchymatous  organs,  such 
as  the  kidneys,  heart,  and  liver,  causing  coagulation  of  the  protoplasm 
(cloudy  swelling)  and  final  dissolution.  The  harm  brought  about  by 
the  toxins  or  toxic  products  of  the  pyogenic  group  of  microorganisms, 
for  instance,  acts  mainly  in  this  manner. 

SPECIAL  PROPERTIES  OF  THE  PRINCIPAL  TOXINS 
1.  Diphtheria  Toxin. — Diphtheria  bacilli  vary  considerably,  both  in 
tissues  and  in  artificial  culture  media,   in  the   quantity  of   toxin   se- 
creted;   thus  in  bouillon  large  amounts  are  seldom  found  in  less  than 
from  seven  to  fourteen  days. 

The  action  of  the  toxin  is  dependent  upon  the  dosage,  and  a  certain 
period  of  time  must  always  elapse  before  the  symptoms  appear,  the 
minimum  being  about  one  day.  Large  doses  may  shorten  this  period 
of  incubation,  but  cannot  diminish  it  below  a  certain  limit. 

The  lesion  of  diphtheria  is  practically  always  local,  and  is  usually 
situated  on  the  mucous  membrane  of  the  upper  air-passages.  It  is 
characterized  by  the  formation  of  a  pearly  white  membrane  that  is 
adherent  to  the  underlying  edematous  tissues.  The  toxin  produces 
necrosis  of  the  surface  epithelium,  and  the  product,  together  with  fibrin 


114  INFECTION 

and  leukocytes,  constitutes  the  membranous  exudate.  From  this  focus 
toxin  is  absorbed  by  the  lymphatics  and  blood-stream,  and  distributed 
throughout  the  body,  the  bacilli  being  rarely  found  in  the  blood  or  in- 
ternal organs.  Later  the  effects  of  toxin  intoxication  are  shown  by 
paralyses  of  certain  motor  nerves  and  ganglia,  particularly  those  of  the 
palate  and  heart. 

When  a  guinea-pig  receives  a  subcutaneous  inoculation  with  diph- 
theria toxin,  a  typical  hemorrhagic  gelatinous  edema  develops  at  the 
site  of  inoculation  (Fig.  36).  Upon  opening  the  abdominal  cavity  one 
finds  but  little  peritoneal  exudate,  but  the  vessels  of  the  mesentery  are 
injected  and  the  adrenal  glands  show  characteristic  acute  hyperemia  (Figs. 
37  and  38).  Bloody  pericardial  and  pleural  exudates  will  be  found  in  the 
thorax,  and  solidified  areas  in  the  lungs.  Guinea-pigs  surviving  a  dose  of 
toxin  may,  after  two  or  four  weeks,  begin  to  show  paralysis  of  the  hind 
and  then  of  the  fore  extremities,  a  condition  analogous  to  the  post-diph- 
theric paralysis  occurring  in  man  and  ascribed  to  the  effects  of  toxon. 

Method  of  Testing  the  Virulence  and  Toxicity  of  Diphtheria  Bacilli. — 
Young  guinea-pigs  weighing  from  250  to  300  grams  are  quite  susceptible 
to  diphtheria  toxin,  and  are  used  in  determining  the  strength  of  a  toxin 
and  in  standardizing  antitoxin.  The  test  may  be  of  great  value  in  the 
management  of  convalescent  and  " carrier"  cases  of  diphtheria,  harbor- 
ing bacilli  in  the  upper  air-passages,  in  determining  whether  the 
microorganisms  are  dangerous  or  merely  harmless  non-pathogenic  sapro- 
phytes. It  is  practically  impossible,  from  the  morphology  of  the  organ- 
ism alone,  to  decide  whether  or  not  a  given  culture  is  dangerous,  and 
prolonged  quarantine  may  not  only  be  irksome  and  inconvenient,  but, 
if  the  organisms  are  proved  to  be  harmless,  it  is  unnecessary  as  well. 

To  be  reliable,  however,  such  a  test  must  be  carried  out  very  care- 
fully. In  the  case  of  a  highly  virulent  culture,  the  mere  introduction 
of  a  few  organisms  beneath  the  skin  will  suffice  to  demonstrate  their 
dangerous  character,  but  with  cultures  only  slightly  virulent,  more  care 
is  necessary,  for  although  the  patient  may  show  no  ill  effects  as  a  result 
of  the  presence  of  the  bacilli,  in  the  throat  of  another  and  less  immune 
individual  they  may  be  highly  dangerous. 

The  following  method  has  been  used  by  the  author  in  many  hundreds 
of  such  tests  in  the  Philadelphia  Hospital  for  Contagious  Diseases,  and 
has  proved  of  distinct  value : 

1.  Make  a  culture  of  the  part  harboring  the  bacilli  on  a  tube  of  Loffler  serum 
medium.  Incubate  at  35°  C.  for  from  eighteen  to  twenty-four  hours;  prepare  a 
smear  and  stain  with  Loffler's  methylene-blue.  If  diphtheria  bacilli  are  present, 


FIG.  36.— ABDOMINAL  WALL  OF  GUINEA-PIG  SHOWING  DIPHTHERIC  EDEMA. 
Shows  abdominal  wall  of  a  guinea-pig  forty-eight  hours  after  subcutaneous  in- 
jection with  2  c.c.  of  a  seventy-two-hour  bouillon  culture  of  a  diphtheria  bacillus 
isolated  from  the  throat  of  a  diphtheria  convalescent. 


FIG.  37. — NORMAL  ADRENAL  GLAND  OF  A  GUINEA-PIG. 


FIG.  38. — ADRENAL    GLAND    OF    A    GUINEA-PIG    AFTER    FATAL    DIPHTHERIC 

INTOXICATION. 


SPECIAL   PROPERTIES   OF   THE   PRINCIPAL   TOXINS  115 

they  must  be  isolated  in  pure  culture.     Never  attempt  a  guinea-pig  test  with  an 
impure  culture! 

2.  Isolate  by  the  "streak"  method,  on  plates  of  blood-serum. 

3.  Inoculate  a  tube  of  1  per  cent,  glucose  bouillon,  which  is  neutral  or  slightly 
alkaline,  with  several  different  colonies. 

4.  Incubate  at  35°  C.  for  three  days,  keeping  the  tube  in  a  slanted  position  in 
order  to  give  the  culture  as  much  oxygen  as  possible.     If  a  good  growth  does  not 
appear  in  twenty-four  hours,  transplant  to  another  tube  of  bouillon  until  the  bacilli 
have  been  "educated"  to  grow  on  the  medium. 

5.  Examine  for  purity.     Select  a  250-  to  300-gram  guinea-pig  and  inject  2  c.c. 
of  the  unfiltered  culture  in  the  median  abdominal  line.     Animals  over  the  weight 
specified  are  more  resistant  and  less  reliable  for  the  test.     The  unfiltered  culture  is 
used,  since  toxin  is  but  one  element  of  the  disease-producing  power  of  diphtheria 
bacilli,  and  toxin  production  in  bouillon  may  not  be  a  true  index  of  the  toxin  produc- 
tion in  mucous  membranes. 

6.  Carefully  observe  the  animal  for  at  least  four  days.     Even  slight  toxemia, 
especially  if  accompanied  by  edema  at  the  site  of  injection,  should  be  regarded  as  a 
positive  result  (Fig.  37). 

7.  After  death  perform  a  careful  autopsy.     Make  cultures  of  the  edematous 
area,  peritoneum,  and  heart  blood.     Diphtheria  bacilli  may  be  found  in  the  edema- 
tous fluid,  but  will  rarely  be  found  in  the  peritoneum  or  in  the  blood.    Observe  whether 
acute  hyperemia  of  the  suprarenal  glands  is  present  (Figs.  37  and  38). 

8.  Not  infrequently  animds  showing  mild  or  even  an  absence  of  the  symptoms 
of  toxemia  develop  paralysis  cf  the  hind  quarters  two  or  three  weeks  later.     Accord- 
ing to  Ehrlich,  this  paralysis  is  due  to  the  action  of  "toxon, "  a  toxic  substance  se- 
creted by  the  bacillus  or,  as  believed  by  others,  a  modified  form  of  toxin. 

9.  To  prove  that  diphtheria  was  the  cause  of  the  toxemia  or  death  mix  2  c.c.  of 
the  culture  in  a  test-tube  with  1  c.c.  of  diphtheria  antitoxin  (500  units).     After  stand- 
ing aside  for  an  hour  at  room  temperature,  inject  the  mixture  subcutaneously  in  the 
median  abdominal  line  of  a  250  to  300  gram  guinea-pig.     Symptoms  of  toxemia  do 
not  develop. 

In  a  comparative  study  of  the  above  and  other  methods  Kolmer 
and  Moshage1  found  that  washing  off  a  pure  culture  from  a  slant  of 
Loeffler's  blood-serum  media  with  10  c.c.  of  sterile  salt  solution,  emulsi- 
fying and  injecting  4  c.c.  subcutaneously  in  the  medium  abdominal  line 
of  a  250-  to  300-gram  guinea-pig,  yielded  equally  delicate  results.  The 
intracutaneous  method  of  Neisser  which  has  also  been  advocated  by 
Zingher  and  Soletsky,2  while  being  more  economical  in  that  2  pigs  suffice 
for  4  or  even  6  tests,  was  found  to  yield  somewhat  indefinite  reactions 
with  bacilli  of  low  virulence. 

Standardizing  Diphtheria  Toxin. — The  strength  of  a  diphtheria  toxin 
is  estimated  by  injecting  subcutaneously  a  series  of  guinea-pigs  weighing 
approximately  250  grams,  with  decreasing  amounts  of  toxin.  How 
many  dilutions  will  be  necessary  it  is  impossible  to  state;  for  exact  results 
several  pigs  of  the  same  weight  should  be  inoculated  with  the  same  dose, 
and  the  effects  should  show  various  gradations,  dependent  upon  the 

1  Jour.  Infect.  Dis.,  1916,  19,  1.  2  Jour.  Infect.  Dis.,  .1915,  17,  454. 


116  INFECTION 

size  of  the  successive  doses.  In  order  to  obtain  a  uniform  method  for 
estimating  the  strength  of  a  diphtheria  toxin  and  thus  obtain  compara- 
tive values,  a  standard  unit  has  been  adopted,  consisting  of  the  smallest 
amount  of  toxin  that  will  kill  a  healthy  guinea-pig  weighing  about  250 
grams  in  from  four  to  five  days.  This  is  known  as  the  minimum  lethal 
dose,  or  dosis  lethalis  minimus.  The  technic  used  for  determining  this 
dose  is  given  in  the  chapter  on  Antitoxins,  p.  241. 

A  quick  and  accurate  method  for  estimating  the  amount  of  diph- 
theria toxin  present  in  the  body-fluids  of  a  diphtheric  patient  would  be 
of  value  in  controlling  the  antitoxin  treatment  of  this  infection.  At 
present  the  amount  of  antitoxin  administered  is  regulated  according  to 
the  clinical  condition  of  the  patient.  Uffenheimer  has  used  a  method 
for  determining  the  presence  of  toxin,  consisting  in  injecting  intra- 
peritoneally  a  250-gram  guinea-pig  with  0.1  to  0.4  c.c.  of  the  patient's 
serum,  diluted  with  2  to  4  c.c.  of  salt  solution.  The  presence  of  a  dis- 
tinct doughy  edema  of  the  abdominal  cavity  after  seventeen  to  twenty- 
four  hours  indicates  the  presence  of  diphtheria  toxin,  an  observation  that 
may  be  confirmed  by  making  an  autopsy  at  the  end  of  forty-eight  hours. 
The  diagnostic  value  of  this  method  has  not  been  adequately  established : 
it  is  doubtful  if  it  yields  any  information  other  than  is  more  readily 
gained  by  making  a  good  cultural  examination  of  the  patient,  and  it 
does  not  aid  in  the  estimation  of  the  quantity  of  toxin,  which  is  the  result 
most  desired. 

Diphtheria  toxins  have  been  classified  into  three  groups,  depending 
upon  the  degree  of  avidity  for  antitoxin  they  display,  viz.,  prototoxin, 
deuterotoxin,  and  tritotoxin.  Each  of  these  toxin  groups  may,  in 
whole  or  in  part,  be  converted  into  toxoids.  The  prototoxin  has  a 
greater  affinity  for  the  antitoxin  than  has  the  deuterotoxin,  and  the 
deuterotoxin  has  a  greater  affinity  for  the  antitoxin  than  has  the  trito- 
toxin. The  same  relation  is  apparent  with  the  three  toxoids,  which  are 
not  poisonous,  but  which  have  the  same  power  of  combining  with 
antitoxin  as  have  the  toxins  from  which  they  take  their  origin. 

In  standardizing  antitoxin,  it  is  found  in  general  that  with  a  perfectly 
fresh  toxin  a  certain  amount  of  antitoxin  will  just  neutralize  a  definite 
amount  of  toxin.  If  older  toxin  is  used,  it  is  found  that  the  toxin  has 
lost  about  one-half  its  toxic  power,  but  retains  its  initial  power  for 
neutralizing  antitoxin.  Ehrlich  explained  this  by  showing  that  the 
diphtheria  toxin  molecule  is  composed  of  two  groups — one  the  carrier 
of  the  toxic  qualities,  the  toxophore  group,  which  is  quite  labile;  the 
other  uniting  the  whole  molecule  with  antitoxin,  being  capable  of  neu- 


SPECIAL    PROPERTIES    OF   THE    PRINCIPAL   TOXINS  117 

tralizing  it,  and  characterized  by  its  stability.  The  toxophore  group 
being  destroyed  as  in  old  toxin,  the  poison  loses  its  toxic  qualities,  birt 
retains  its  power  to  bind  antitoxin.  This  modified  toxin  or  non-poisonous 
diphtheria  toxin  has  been  designated  by  Ehrlich  "diphtheria  toxoid." 

2.  Tetanus  Toxin. — Of  all  bacteria  classed  as  true  toxin  producers, 
none  possesses  greater  toxicity  than  does  the  tetanus  bacillus.     The 
number  of  organisms  producing  sufficient  toxin  to  cause  a  fatal  infection 
may  be  so  small  that  careful  anaerobic  cultures  made  from  the  local 
lesion  of  infection,  together  with  injection  of  the  wound  secretions  into 
white  mice,  may  fail  to  disclose  the  presence  of  tetanus  bacilli. 

According  to  Ehrlich,  tetanus  toxin  is  composed  of  two  separate  and 
distinct  substances — (1)  Tetanospasmin,  a  neurotoxin,  which  is  very 
labile  and  responsible  for  the  severe  symptoms  of  the  infection;  (2) 
tetanolysin,  a  hemotoxin,  which  is  more  stable  and  destructive  for  ery- 
throcytes. 

Tetanus  toxin  is  prepared  by  cultivating  the  bacillus  in  bouillon 
under  strict  anaerobic  conditions.  Since  tetanospasmin  is  so  suscep- 
tible to  the  influence  of  heat,  age,  and  even  light,  the  toxin  is  best  pre- 
served in  a  dry  form.  The  standard  of  tetanus  toxin  consists  of  100 
minimal  lethal  doses  of  a  precipitated  and  dried  toxin,  preserved  at  the 
Hygienic  Laboratory  of  the  Public  Health  and  Marine-Hospital  Service. 

If  susceptible  animals,  such  as  mice  or  guinea-pigs,  are  injected  sub- 
cutaneously  or  intravenously  with  tetanus  toxin,  they  begin  to  manifest 
symptoms  after  a  certain  period;  these  are  due  to  the  action  of  tetano- 
spasmin upon  motor  nerve-cells,  and  are  characterized  by  hyper- 
sensitiveness,  clonic  convulsions,  and  rigidity  of  the  muscles.  In  man 
the  symptoms  of  tetanus  are  similar  to  those  in  the  animal,  the  spasm 
starting  quite  regularly  in  the  muscles  of  the  lower  jaw. 

Experiments  by  Wassermann  and  Takaki  have  demonstrated  that 
an  especially  close  affinity  exists  between  tetanus  toxin  and  certain 
structures,  particularly  that  of  the  central  nervous  system.  Most 
writers  agree  that  the  toxin  reaches  these  tissues  largely  by  way  of  the 
nerve-paths. 

3.  Botulism  Toxin. — This  poison  is  generated  by  the  Bacillus  bot- 
ulinus,  first  isolated,  by  Van  Ermengem  in  1896,  from  a  ham  during  an 
epidemic  of  meat  poisoning.     It  is  the  cause  of  a  type  of  meat  and  sau- 
sage poisoning  called  botulism,  more  frequent  in  those  countries  where 
raw  meat  is  eaten,  and  frequently  confused  with  "ptomain  poisoning." 

The  bacillus  is  a  motile,  spore-forming,  anaerobic  bacterium,  which 


118  INFECTION 

grows  at  room  temperature  and  causes  marked  gas  formation  in  glucose 
media. 

The  toxin  is  readily  produced  in  anaerobic  alkaline  bouillon  cultures. 
It  is  quite  labile. 

Symptoms  of  botulism  appear  only  after  a  definite  period  of  incuba- 
tion, which  varies  from  twenty-four  to  forty-eight  hours.  In  contra- 
distinction to  the  meat  poisonings  produced  by  other  organisms,*  those 
due  to  Bacillus  botulinus  may  show  few  or  no  symptoms  directly  re- 
ferable to  the  intestinal  tract,  the  chief  symptoms  being  due  to  toxic 
interference  with  the  cranial  nerves:  loss  of  accommodation,  ptosis, 
dilated  pupils,  aphonia,  dysphagia,  and  hypersecretion  of  mucus  from 
the  mouth  and  nose. 

Guinea-pigs  are  quite  susceptible,  and  may  be  infected  by  way  of  the 
mouth.  The  symptoms  of  intoxication  usually  follow  in  twenty-four 
hours,  and  are  characterized  by  motor  paralysis,  dyspnea,  and  hyper- 
secretion  of  mucus  from  the  nose  and  mouth. 

4.  Dysentery  Toxin. — The  distinct  types  of  dysentery  bacilli  vary 
exceedingly  in  their  powers  to  produce  toxins,  the  strongest  poisons 
being  produced  with  bacilli  of  the  Shiga-Kruse  variety,  less  regularly 
active  ones,  with  bacilli  of  the  Flexner  type. 

Investigations  have  shown  quite  conclusively  that  dysentery  itself 
is  a  true  toxemia,  its  symptoms  being  referable  to  the  absorption  of  the 
toxins  of  the  bacillus  from  the  intestine.  Flexner,  who  has  studied  this 
subject  with  great  care,  believes  it  probable  that  most  of  the  pathologic 
lesions  occurring  in  the  intestinal  canal  are  referable  to  the  excretion  of 
dysentery  toxin,  rather  than  to  the  direct  local  action  of  the  bacilli. 
The  action  of  the  dysentery  toxin  upon  animals  is  very  characteristic, 
and  throws  much  light  upon  the  disease  in  man.  Intravenous  injection 
of  the  toxin  in  rabbits  is  followed  by  marked  diarrhea,  rapid  fall  in  tem- 
perature, respiratory  embarrassment,  and  terminal  paralysis.  Upon 
autopsy  the  intestinal  mucosa,  especially  that  of  the  cecum  and  colon, 
shows  marked  inflammatory  involvement,  supporting  Flexner's  ob- 
servation of  the  necrotic  action  of  excreted  toxin. 

Dysentery  bacilli  also  produce  an  endotoxin,  and  poisonous  sub- 
stances are  easily  obtained  by  extracting  the  bacilli  themselves  or  by 
filtration  of  properly  prepared  bouillon  cultures.  The  toxin  is  fairly 
stable,  and  well  preserved  under  toluol  in  the  refrigerator. 

5.  Staphylolysin. — Two   definite   toxins   have   been   isolated   from 
cultures  of  Staphylococcus  pyogenes  aureus  and  albus,  one  of  which 


TOXINS    OF   THE    HIGHER    PLANTS    AND    ANIMALS  119 

exerts  a  destructive  action  on  erythrocytes  (hemotoxin),  and  the  other 
on  leukocytes  (leukocidin). 

An  anti-hemotoxin  that  counteracts  the  effects  of  the  toxin  may  be 
produced  experimentally,  and  in  human  staphylococcus  infections  the 
demonstration  of  such  antihemotoxic  substances  in  the  blood-serum 
may  be  of  aid  in  making  the  diagnosis  of  staphylococcus  infections. 
This  antistaphylolysin  may  be  found  normally  in  small  amounts  in  the 
serum  of  man  and  horse,  and  when  anti-hemotoxic  tests  with  human 
serum  are  made,  a  normal  control  should  always  be  included.  Anti- 
leukocidins  have  also  been  produced,  but  are  not  of  practical  im- 
portance. 

The  hemotoxin  is  readily  formed  in  cultures  of  staphylococci; 
roughly,  the  amount  produced  depends  upon  the  virulence  of  the  culture. 
In  human  cases  of  staphylococcus  infections  this  toxin  produces  hemoly- 
sis  in  vivo,  and  is  partly  responsible  for  the  grave  anemia  that  is  fre- 
quently present. 

6.  Streptolysin. — The  grave  systemic  symptoms  that  so  frequently 
accompany  slight  streptococcus  lesions  are  strong  indications  that  these 
microorganisms  produce  a  powerful  diffusible  poison,  although  extensive 
researches  into  the  nature  of  these  poisons  have  not  given  us  any  clear 
understanding  of  the  subject. 

Streptococci  may  yield  soluble  toxins  that,  when  administered  to 
guinea-pigs,  produce  rapid  collapse  and  death.  While  these  toxins  are 
not  comparable  in  potency  to  the  soluble  toxins  of  diphtheria  and 
tetanus,  they  have,  nevertheless,  been  differentiated  from  the  endo- 
toxins  contained  within  the  cell-bodies,  and  have  been  found  to  possess 
less  toxicity. 

Beside  these  toxins,  some  streptococci  produce  a  hemolysin  which 
may  be  conveniently  observed  by  cultivation  of  the  organisms  upon 
blood-agar  plates.  This  hemotoxin  is  partly  responsible  for  the  san- 
guineous character  of  a  streptococcus  exudate. 


TOXINS  OF  THE  HIGHER  PLANTS  AND  ANIMALS 

PHYTOTOXINS 

As  has  previously  been  mentioned,  the  power  of  forming  toxins  is  not 
confined  to  bacteria  alone.  There  is  a  class  of  higher  plants  and  ani- 
mals that  produces  characteristic  poisons  against  which  immunization 
can  be  undertaken  and  an  antitoxic  serum  obtained.  Those  of  most 
interest  medically  are  pollen  toxin  and  snake  poison. 


120  INFECTION 

The  most  important  plant  toxins  (phytotoxins)  are  ricin,  abrin, 
crotin,  and  pollen.  All  possess  more  or  less  toxic  qualities;  the  first 
three  either  agglutinate  or  hemolyze  the  corpuscles  of  certain  animals. 
Antitoxic  serums  have  been  prepared  that  will  neutralize  the  respective 
toxins,  and  this  factor  constitutes  the  most  important  evidence  of  their 
toxin-like  character. 

General  Properties. — These  toxins  resemble  proteins  in  many  re- 
spects. Jacoby,  however,  has  placed  them  in  the  same  class  as  bacterial 
toxins  and  enzymes,  i.  e.,  large  molecular  colloids  closely  resembling  the 
proteins,  but  still  not  giving  the  usual  protein  reaction.  More  recent 
work  by  Osborne,  Mendel,  and  Harris,1  however,  does  not  support 
Jacoby's  view.  These  observers  found  the  toxic  properties  of  ricin 
inseparably  associated  with  the  coagulable  albumin,  and  were  able  to 
isolate  it  in  such  strength  that  10100  milligram  was  fatal  per  kilo  of  rabbit, 
and  solutions  of  0.001  per  cent,  would  agglutinate  red  corpuscles. 

Relation  to  Immunity. — The  phytotoxins,  since  they  obey  the  same 
laws  as  bacterial  toxins,  have  been  very  serviceable  in  the  study  of  im- 
munity; they  are  more  stable  than  the  latter,  and  can  be  handled  in 
more  exact  and  definite  quantities.  They  have  apparently  the  same 
haptophore  and  toxophore  structure  as  bacterial  toxins;  antitoxins  are 
readily  produced  by  immunizing  animals,  and  seem  to  be  specific  for  the 
toxins;  in  fact,  Ehrlich  made  his  earliest  observations  on  the  specificity 
and  quantitative  factors  in  toxin-antitoxin  immunity  from  a  study  of 
these  plant  toxins. 

The  Toxin  of  Hay-fever. — Pollen  toxin  has  been  described  by 
Dunbar2  as  the  etiologic  factor  in  the  production  of  hay-fever.  In  all, 
the  pollen  of  25  varieties  of  grass  and  seven  varieties  of  plants  have  been 
found  capable  of  producing  attacks  of  hay-fever  in  susceptible  persons. 
This  susceptibility  to  pollen  intoxication  is,  fortunately,  limited,  and  the 
sudden  onset  of  an  attack  and  the  characteristic  symptoms  indicate  an 
anaphylactic  reaction  due  to  sensitization  with  pollen  protein.  Dunbar 
has  succeeded  in  producing  a  pollen  antitoxin,  which  will  be  described 
in  the  chapter  on  Passive  Immunization;  the  reports  of  various 
observers  are,  however,  at  variance  in  regard  to  its  therapeutic  value. 

It  is  highty  probable  that  the  toxic  substance  in  ivy,  sumac  and 
other  plants  responsible  for  vario.us  forms  of  dermatitis  venenata,  is  of 
the  nature  of  plant  or  phytotoxin  of  protein  nature. 

1  Amer  Jour.  Physiol.,  1905,  14,  259. 

2  For  full  review  of  this  subject  see  Glegg:  Jour.  Hygiene,  1904,  4;  Liefman: 
Zeit.  f.  Hygiene,  1904,  47,  153;  Wolff-Eisner,  Deut.  med.  Woch.,  1906,  32,  138. 


TOXINS    OF   THE    HIGHER    PLANTS    AND    ANIMALS  121 

ZOOTOXINS 

The  most  important  animal  toxins  (zootoxins)  are  those  of  the  toad, 
spider,  snake,  scorpion,  and  bee.  The  most  striking  characteristic  of 
these  toxins  is  that  an  immunity  against  them  can  be  established;  in 
this  respect  they  resemble  true  toxins.  All  are  quite  complex  in  struc- 
ture and  properties,  and  all  are  more  or  less  hemotoxic. 

Snake  Venoms.1 — Medically,  these  are  of  particular  interest.  They 
were  first  thoroughly  investigated  by  S.  Weir  Mitchell  (1860)  and 
Mitchell  and  Reichert  (1883),  and  have  aroused  considerable  attention 
because  of  their  similarity  to  bacterial  toxins  and  the  aid  their  study 
has  been  in  the  elucidation  of  immunologic  problems. 

Properties  of  Venom. — In  1883  Mitchell  and  Reichert  described  two 
poisonous  proteins,  constituents  of  venom,  one  of  which  seemed  to  be  a 
globulin  and  the  other  a  proteose  or  "peptone."  Faust2  believes  that 
the  poisons  are  not  proteins,  but  glucosids  free  from  nitrogen,  and  that 
they  belong  to  the  saponin  group  of  hemotoxic  agents.  It  may  be  that 
these  glucosids  are  bound  to  proteins,  and  can  be  removed  with  the 
globulin  in  fractional  separation,  or  that  they  may  come  down,  at  least 
in  part,  with  the  albumoses  of  the  venom. 

Various  enzymes  have  been  found  in  venoms;  e.g.,  proteases  (Flex- 
ner  and  Noguchi)  and  Upases  (Noguchi);  the  latter  probably  have  a 
definite  relation  to  many  of  the  effects  of  venom  intoxication,  especially 
hemolysis  and  fatty  degeneration  of  the  tissues. 

The  poisons,  as  a  rule,  produce  both  local  and  severe  general  dis- 
turbances, the  rapidity  of  the  onset  of  the  symptoms  and  the  prognosis 
in  a  given  case  depending  largely  on  the  situation  of  the  bite.  Most  of 
these  poisons  exert  their  effect  primarily  upon  the  nervous  and  vascular 
systems,  besides  exhibiting  other  toxic  properties. 

Nature  of  Venoms. — All  snake  venoms  possess  a  hemolytic  power,  and 
venom  hemolysis  is  one  of  the  most  interesting  of  biologic  phenomena. 
Flexner  and  Noguchi8  have  distinguished  and  classified  the  various 
elements  as  hemotoxins,  hemagglutinins,  neurotoxins,  leukotoxins,  and 
endotheliotoxins  (hemorrhagin).  The  endotheliolytic  action  of  the 
toxins  is  shown  in  the  glomerular  capillaries,  where  it  causes  hemorrhage 
and  hematuria  (Pearce4). 

1  See  Faust:  "Die  tierischen  Gifte,"  Braunschweig,  1906;  Noguchi:  Carnegie 
Institution   Publications,    1909,   No.    Ill;    Calmette:    "Les  venins,"   etc.,   Paris, 
Masson,  1907. 

2  Arch.  exp.  Path.  u.  Pharm.,  1907,  56,  236;  1911,  64,  244. 

3  Jour.  Exp.  Med.,  1903,  9,  257;  Univ.  of  Penna.  Med.  Bull.,  1902,  15,  345. 

4  Jour.  Exp.  Med.,  1909,  11,  532. 


122  INFECTION 

Cobra  hemotoxin  is  especially  characterized  by  its  power  of  dissolv- 
ing the  corpuscles  of  certain  species  (man,  dog,  guinea-pig,  rabbit)  with- 
out the  presence  of  serum.  The  explanation  of  this  interesting  phenome- 
non has  excited  extensive  discussion.  It  is  probable  that  the  hemotoxin 
is  in  the  nature  of  an  amboceptor  (Flexner  and  Noguchi),  which  is 
activated,  in  the  absence  of  serum,  by  complementing  substances  (chiefly 
lecithin)  present  in  the  red  cells,  and  in  this  manner  producing  hemolysis 
of  these  cells.  In  syphilis  the  quantity  of  red-cell  lecithin  is  probably 
diminished  after  the  primary  stage,  so  that  when  using  definite  dilu- 
tions of  venom  that  are  known  to  hemolyze  a  certain  quantity  of  normal 
erythrocytes,  an  absence  of  hemolysis  of  the  red  corpuscles  of  a  given 
patient  would  infer  a  decrease  in  complementing  lecithin  in  these  cor- 
puscles and  indicate  the  presence  of  syphilis.  The  technic  of  this  reac- 
tion and  its  value  as  a  diagnostic  procedure  will  be  discussed  further  on 
under  the  head  of  Venom  Hemolysis. 

ENDOTOXINS 

There  are  a  large  number  of  microorganisms,  notably  the  cholera 
spirillum,  the  typhoid  bacillus,  the  pneumococcus,  and  other  pyogenic 
cocci,  which,  when  cultivated  and  separated  from  the  culture-fluid  by 
filtration,  are  found  to  be  highly  poisonous,  whereas  the  filtrate  itself 
is  practically  devoid  of  toxicity  except  for  the  soluble  hemotoxic  sub- 
stances. In  other  words,  we  are  dealing  with  endotoxins,  or  poisons  that 
are  not  secreted  into  the  medium  in  which  the  bacteria  are  growing,  but  are 
contained  more  or  less  firmly  within  the  bacterial  body,  from  which  they 
are  separable  by  some  method  of  extraction  or  by  autolysis,  only  after 
death. 

Method  of  Obtaining  Endotoxic  Substances. — Endotoxic  substances 
are  obtained  from  bacteria  by  thorough  disintegration  of  the  bodies. 
This  is  accomplished  by  various  methods:  (a)  The  substances  may  be 
found  in  old  cultures  as  a  result  of  death  and  disintegration  of  numbers 
of  bacteria;  (6)  they  may  be  obtained  by  suspending  the  microorganisms 
in  distilled  water  and  shaking  in  a  machine,  much  as  Wassermann  and 
Citron's  artificial  "aggressins"  are  prepared;  (c)  the  bacteria  may  be 
dried  and  ground  to  a  fine  powder,  as  in  the  preparation  of  Koch's 
"bacillen  emulsion"  of  tubercle  bacilli;  (d)  MacFadyen  freezes  masses  of 
bacteria  with  liquid  air,  and  then  grinds  them  into  a  fine  powder;  (e) 
Conradi  recommends  autolyzing  the  bacterial  cells  in  non-nutrient 
fluids;  (/)  Rosenau  has  studied  the  endotoxins  of  pneumococci  obtained 
by  alternate  freezing  and  thawing  of  suspensions  in  distilled  water;  (g) 


ENDOTOXINS  123 

Vaughan  has  devised  a  method  of  growing  massive  cultures  on  solid 
media  several  square  yards  in  extent,  removing  the  bacteria  with  sterile 
salt  solution,  and  digesting  the  bacterial  masses  with  an  excess  of  a  2 
per  cent,  solution  of  caustic  alkali  in  absolute  alcohol. 

In  the  body,  the  endotoxins  are  probably  liberated  either  by  autolysis 
or  by  disintegration  through  the  bacteriolysins,  complements,  or  enzymes 
of  the  body-cells  and  fluids. 

Nature  of  Endotoxins. — Owing  to  their  insoluble  nature,  endotoxins 
in  pure  form  and  free  from  other  products  of  bacterial  activity,  cannot 
be  obtained.  As  a  result,  their  chemical  nature  and  structure  are  un- 
known. Tuberculin,  which  was  formerly  believed  to  be  an  albumose, 
may  be  produced  in  a  protein-free  medium;  it  seems  probable  that  this 
substance  is  of  the  nature  of  a  polypeptid,  giving  no  biuret  reaction,  but 
being  destroyed  by  pepsin  and  trypsin  (Laevenstein  and  Pick  J).  Whether 
or  not  tuberculin  is  an  endotoxin  liberated  upon  the  disintegration  of  the 
bacilli  is  unknown.  Pick  regards  it  as  a  secretory  product  closely 
related  to  the  true  toxins.  It  is  probable  that  some  toxin  is  actually 
secreted  into  the  culture-medium,  and  that  the  major  portion,  which  is 
of  a  somewhat  different  nature,  is  intimately  related  to  the  constituents 
of  the  bacterial  cells. 

There  is  little  doubt  but  that  endotoxic  substances  are  highly  poison- 
ous, and  that  they  are  chiefly  responsible  for  the  characteristic  symptoms 
of  diseases  produced  by  the  bacteria  that  contain  them.  Whether, 
however,  they  are  actually  preformed  definite  and  specific  constituents 
of  bacteria,  or  merely  the  poisonous  products  of  disintegration  of  the 
bacterial  proteins,  is  still  undecided.  It  would  appear  that  bacteria 
produce  and  contain  toxic  substances.  When,  owing  to  the  peculiar 
structure  of  the  bacterial  protoplasm  or  nature  of  the  toxic  substance 
itself,  the  toxin  can  diffuse  readily  into  a  surrounding  medium,  the  toxic 
substance  is  known  as  a  true,  soluble,  or  extracellular  toxin;  when  the 
toxin  enters  into  combination  with  the  bacterial  protoplasm,  it  becomes 
known  as  an  endotoxin.  This  union  of  toxin  and  bacterial  protoplasm 
may  be  so  firm  as  to  render  the  toxic  substance  inseparable  from  the 
bacterial  protein.  The  various  toxic  substances  or  toxins  differ,  there- 
fore, according  to  their  diffusibility  through  the  membrane  of  bacterial 
protoplasm,  or  their  power  of  combining  with  these  protein  substances, 
or  both  factors  may  be  operative. 

Satisfactory  antitoxins  for  endotoxins  have  not  been  produced,  and  this 
is  an  important  point  in  differentiating  between  a  true  toxin  or  an  endotoxin 
of  any  particular  microorganism.  Animals  immunized  against  endotoxin 
^iochem.'Zeit.,  1911,  31,  142. 


124  INFECTION 

develop  substances  in  their  serum  that  are  bactericidal,  bacteriotropic, 
and  agglutinative  to  the  bacteria  from  which  the  poisons  were  derived, 
but  the  serum  itself  is  not  antitoxic  for  the  endotoxins.  Therapeutic 
serums  for  use  against  infections  caused  by  the  endotoxin  class  of 
bacteria  are  largely  bacteriolytic  and  bacteriotropic  in  action.  The 
endotoxins  of  some  bacteria,  and  particularly  those  of  streptococci, 
seem  to  repel  the  leukocytes,  or  exert  a  negative  chemotactic  influence, 
which  may  effectually  retard  or  entirely  prevent  phagocytosis;  in  this 
respect  they  resemble  the  aggressins  of  Bail.  Immune  serums  owe  a 
portion,  at  least,  of  their  therapeutic  value  to  the  power  they  possess  of 
overcoming  this  influence  and  facilitating  phagocytosis.  These  serums, 
however,  have  not  proved  of  as  much  value  as  have  the  diphtheria  and 
tetanus  antitoxins  in  the  treatment  of  the  respective  infections  men- 
tioned, and  have  proved  a  check  to  the  progress  of  serum  therapy.  It 
is  probable  that  the  endotoxins  are  more  specific  for  the  various  strains 
of  the  same  species  than  are  the  true  toxins,  as  indicated  by  the  results 
of  Cole  in  the  treatment  of  pneumonia  with  an  anti-pneumococcus 
serum  corresponding  to  the  type  of  microorganism  responsible  for  the 
individual  infection,  as  determined  by  a  rapid  method  of  diagnosis 
previous  to  the  administration  of  serum. 

AGGRESSINS 

In  an  attempt  to  explain  certain  observations  of  Koch  to  the  effect 
that  when  a  tuberculous  animal  is  injected  intraperitoneally  with  a  fresh 
culture  of  tubercle  bacilli  it  succumbs  quickly  to  an  acute  attf»<^  ^  the 
disease,  the  resulting  exudate  being  composed  almost  exclusively  of 
lymphocytes,  Bail1  has  advanced  the  hypothesis  that  bacteria  may 
secrete  aggressins,  or  substances  that  aim  to  protect  the  microorganism 
by  either  neutralizing  the  action  of  opsonins  or  directly  repelling  the 
body-cells  and  preventing  phagocytosis.  Bail  found  that  if  he  removed  a 
tuberculous  exudate,  sterilized  it,  and  injected  it  into  healthy  animals, 
it  had  practically  no  effect.  If  tubercle  bacilli  were  injected  alone, 
lesions  would  develop  in  the  usual  number  of  weeks;  but  if  sterile 
exudate  and  tubercle  bacilli  were  injected  together,  death  would  follow 
in  about  twenty-four  hours,  indicating  that  the  exudate  contained  a 
substance  that  acutely  paralyzed  the  defensive  forces  of  the  animal,  and 
thus  greatly  increased  the  virulence  of  the  bacilli.  That  this  effect  was 
not  the  summation  of  endotoxins  in  the  exudate  plus  living  microorgan- 

1Wien.  klin.  Woch.,  1905,  8,  14,  16,  and  17;  Berl.  klin.  Woch.,  1905,  15;  Zeit. 
f.  Hyg.,  1905,  i/A3;  Arch.  f.  Hyg.,  1905,  52,  272,  and  411. 


AGGRESSINS  125 

isms  was  shown  by  Bail,  who  found  that  when  large  quantities  of  exudate 
alone  were  injected  no  untoward  effects  resulted,  whereas  the  injection 
of  a  small  amount  of  exudate,  plus  a  sublethal  dose  of  bacteria,  would 
regularly  produce  acute  infection  and  death.  Bail  therefore  concluded 
that  the  exudate  contained  a  substance  that  allowed  the  bacilli  to  become 
more  aggressive,  and  for  this  reason  he  called  this  hypothetic  substance 
"aggressin. "  He  assumes  that  in  a  tuberculous  animal  the  tissues  are 
permeated  with  the  aggressin,  and  that  when  fluid  collects  in  the  body- 
cavities  after  the  injection  of  tubercle  bacilli,  this  fluid  contains  large 
quantities  of  aggressin.  This  prevents  migration  and  collection  of 
polynuclear  leukocytes,  but  not  of  lymphocytes,  and  hence  allows  the 
bacilli  to  develop  rapidly,  producing  acute  symptoms.  On  the  other 
hand,  when  tubercle  bacilli  are  injected  into  the  peritoneal  cavity  of 
a  healthy  guinea-pig,  polynuclear  leukocytes  which  engulf  the  bacilli 
are  attracted,  thus  inhibiting  their  rapid  development,  there  being  no 
aggressin  to  prevent  phagocytosis. 

Similar  results  were  obtained  with  other  microorganisms.  Bail 
inoculated  cholera  and  typhoid  bacilli  into  the  pleural  and  peritoneal 
cavities  of  animals,  and  an  acute  local  infection  occurred.  From  the 
exudates  so  produced  he  removed  the  bacteria  by  centrifugalization, 
and  completed  the  sterilization  with  antiseptics  or  with  heat  at  44°  C. 
The  clear  fluid  obtained  was  found  to  possess  but  mild  toxic  properties, 
and  large  amounts  could  be  injected  into  animals  of  the  same  species 
without  producing  any  marked  effects;  when,  however,  it  was  injected 
into  an  animal  together  with  a  sublethal  dose  of  the  particular  micro- 
organism, an  acute  and  fatal  infection  followed.  Similar  results  were 
secured  with  the  bacilli  of  dysentery,  chicken  cholera,  pneumonia,  and 
other  diseases. 

Bail's  Classification  of  Bacteria. — Bail  found  that  bacteria  differed 
in  their  power  of  forming  aggressins;  he  therefore  used  this  principle 
in  making  a  division  of  bacteria  into  three  classes,  according  to  their 
disease-producing  power,  as  dependent  largely  upon  whether  or  not  the 
microorganism  can  produce  an  aggressin  that  is  active  against  the  pro- 
tective forces  of  the  host,  particularly  against  opsonins  and  leukocytes. 

1.  Saprophytes,  or  those  bacteria  that,  when  injected  even  in  large 
doses,  do  not  produce  any  characteristic  disease. 

2.  True  parasites,  or  those  bacteria  that,  when  injected  even  in  the 
smallest  amounts,  will  produce  disease  and  death.     These  are  truly 
virulent,  and  the  number  of  bacteria  increase  so  rapidly  as  to  be  demon- 
strable in  every  drop  of  blood  and  in  all  the  organs.     Examples  of  true 
parasites  are  the  bacilli  of  anthrax  and  of  chicken  cholera,  the  tubercle 


126  INFECTION 

bacillus  for  guinea-pigs,  and  the  bacilli  of  the  group  of  hemorrhagic 
septicemia  for  rabbits. 

3.  Half  or  partial  parasites  are  those  bacteria  the  infectious  nature 
of  which  depends  upon  the  number  of  bacteria  injected.  The  smaller 
the  number,  the  milder  the  sjTnptoms,  until  a  dose  is  reached  below 
which  no  disturbances  are  produced.  Organisms  of  this  class  possess 
some  virulence  and  toxicity,  examples  being  the  Bacillus  typhosus  and 
the  Spirillum  cholerae. 

It  is  to  be  remembered,  however,  that  these  effects  are  but  relative, 
and  dependent  upon  the  organism,  the  species  of  animal,  and  the  mode  of 
infection.  For  example,  the  bacillus  of  anthrax  is  saprophytic  for  the 
frog  and  hen  unless  the  temperature  of  these  animals  is  brought  to  the 
body  temperature  of  the  human;  a  bacillus  of  the  group  of  hemorrhagic 
septicemia  of  rabbits  is  saprophytic  for  human  beings,  a  half  parasite 
for  the  guinea-pig  if  injected  subcutaneously,  and  a  true  parasite  for  the 
same  animal  if  injected  intraperitoneally. 

Nature  of  Aggressins. — The  aggressins  in  inflammatory  exudates 
are  presumably  substances  capable  of  paralyzing  the  protective  agencies 
of  the  body.  Bail  regards  the  aggressins  as  of  the  nature  of  endotoxins 
liberated  from  the  bacteria  as  a  result  of  bacteriolysis,  and  believes  that 
they  act  by  paralyzing  the  polynuclear  leukocytes,  thereby  preventing 
phagocytosis.  In  general,  the  production  of  these  aggressins  goes  on 
more  actively  the  greater  the  resistance  to  the  bacteria;  they  are  pro- 
duced in  greater  quantities  during  the  struggle  between  the  bacteria  and 
the  body-cells,  although  they  may  be  produced  artificially  in  the  test- 
tube  with  large  numbers  of  bacteria  and  a  non-poisonous  agent  (serum 
or  distilled  water)  which  can  disintegrate  the  cells.  In  this  manner 
Wassermann  and  Citron  have  produced  "artificial  aggressins, "  which  act 
in  the  same  general  manner  as  the  "natural  aggressins"  of  Bail. 

By  many  the  aggressins  are  regarded  as  endotoxins,  and  while  they 
may  possess  the  nature  of  endotoxic  substances,  it  is  to  be  remembered 
that  there  is  no  definite  relation  between  the  poisonous  qualities  of  the 
aggressins  and  their  power  to  increase  the  virulence  of  an  infection.  It 
is  probable,  as  has  been  shown  by  Wassermann  and  Citron,  that  patho- 
genic bacteria  contain  small  amounts  of  natural  aggressin.  This  ag- 
gressin  may  be  regarded  as  a  normal  antibody  of  the  bacterium  against 
the  defensive  forces  of  the  body-cells  of  a  host.  During  infection  these 
aggressins  or  antibodies  are  naturally  greatly  increased,  as  the  bacteria 
require  more  and  more  protection.  Being  contained  to  some  extent 
within  the  bacterial  cells,  the  antibodies  are  somewhat  similar  to  endo- 
toxins: while  endotoxins  may  be  regarded  as  offensive  agents  of  bacte- 


BACTERIAL    PROTEINS 


127 


ria,  aggressins  may  be  their  defensive  agents.  This  belief  is  in  keeping 
with  the  hypothesis  of  Welch1  and  also  of  Walker2,  according  to  which 
it  may  be  presumed  that  bacteria,  as  living  cells,  when  so  placed  that 
they  are  exposed  to  the  defensive  forces  of  their  host,  are,  under  favor- 
able conditions  stimulated  to  produce  reciprocal  antibodies  for  their 
protection,  and  to  generate  them  in  increasing  amounts  as  may  be  neces- 
sary. 

Bail  regards  the  aggressins  as  new  substances;  as  already  stated  others 
regard  them  as  simple  endotoxins;  still  others  believe  them  to  be  free 
bacterial  receptors,  and  that  these  receptors  may  combine  with  bacterio- 
lytic  amboceptors,  producing,  as  it  were,  a  deflection  of  the  amboceptors,  - 
so  that  the  bacteria  themselves  are  not  attacked,  and  thus  continue  to 
proliferate.  The  action  of  aggressins  is  not  dependent  upon  the  toxicity 
of  the  endotoxins,  for  the  fluid  containing  them  is  devoid  of  toxic  effects; 
at  most,  therefore,  if  they  are  of  the  nature  of  receptors,  they  possess  no 
toxophorous  portion. 

Whatever  aggressins  may  be,  and  we  regard  them  as  antibodies  of 
bacteria,  just  as  bacteriolysins  are  antibodies  of  tissue-cells,  they  appear 
to  be  especially  directed  against  opsonins.  neutralizing  these,  paralyzing 
leukocytes,  and  thus  inhibiting  or  entirely  preventing  phagocytosis. 

Anti-aggressins  may  be  produced  experimentally  by  gradually 
immunizing  animals  with  sterile  exudates,  and  this  immunity  may  be 
transferred  passively  from  one  animal  to  the  other  by  inoculation  of  its 
immune  serum.  These  anti-aggressins  are  quite  specific,  and  neutralize 
the  aggressins  in  an  exudate. 


BACTERIAL  PROTEINS 

In  practically  all  bacterial  bodies,  after  removal  of  toxins  and  endo- 
toxins, a  certain  proteid  residue  remains,  which,  when  injected  into 
animals,  is  able  to  produce  various  grades  of  inflammatory  reaction 
leading  to  tissue  necrosis  and  abscess  formation.  This  substance  was 
first  thoroughly  studied  by  Buchner,  who  named  it  bacterial  protein,  and 
regarded  it  as  identical  in  all  bacteria,  and  having  no  specific  toxic  action, 
but  characterized  in  general  by  its  power  of  exerting  a  positive  chemo- 
tactic  influence  on  leukocytes,  and  thereby  favoring  the  formation  of  pus. 
For  example,  in  the  development  of  an  ordinary  staphylococcus  abscess 
it  is  probable  that  the  proteins  of  the  cocci,  aside  from  their  toxins,  aid 
in  producing  tissue  necrosis  and  in  attracting  leukocytes  to  the  infected 
area.  Similarly,  an  extract  of  dead  tubercle  bacilli  may  produce  a 
it.  Med.  Jour.,  1902,  2,  1105.  2Jour.  of  Path.,  1902,  8,  34. 


128  INFECTION 

tuberculoma  or  the  tissue  changes  incident  to  tuberculosis,  differing, 
however,  from  true  tubercle  in  that  they  do  not  contain  living  bacilli 
and  consequently  are  not  infectious.  When  cultures  of  diphtheria 
bacilli  are  filtered  and  the  residue  washed,  it  is  found  that  extracts  of  the 
bacterial  substances  or  the  bodies  of  the  dead  bacilli  themselves  are  quite 
free  from  the  typical  toxin;  but  the  bacterial  substances  or  the  proteins 
isolated  from  them,  when  injected  into  the  subcutaneous  tissues  of 
animals,  are  found  to  produce  a  strong  inflammatory  reaction  and  necro- 
sis of  the  tissue-cells. 

Bacterial  Split  Proteins. — These  have  been  studied  extensively  by 
Vaughan  and  his  coworkers,  who  have  ascribed  to  them  the  chief  role 
in  and  a  very  important  relation  to  the  processes  of  infection  and  im- 
munity. 

Massive  cultures  of  colon,  typhoid,  pneumonia,  and  diphtheria  microorganisms 
are  grown  in  special  large  tanks  containing  agar;  anthrax  is  grown  in  Roux  flasks, 
and  tubercle  bacilli  in  glycerin  beef-tea  cultures.  After  removal  of  the  growths  the 
bacterial  cellular  substances  are  washed  once  or  twice  with  sterile  salt  solution  by 
decantation,  and  then  repeatedly  washed  with  alcohol,  beginning  with  50  per  cent, 
and  increasing  the  strength  to  95  per  cent.  The  substance  is  then  placed  in  large 
Soxhelet's  flasks  and  extracted  first  for  one  or  two  days  with  absolute  alcohol,  and 
then  for  three  or  four  days  with  ether.  These  extractions  should  be  thorough  in 
order  to  remove  all  traces  of  fats  and  waxes. 

After  extraction  the  cellular  substance  is  ground,  first  in  porcelain,  then  in  agate 
mortars,  and  passed  through  the  finest  meshed  sieves  to  remove  bits  of  agar.  The 
person  grinding  the  cellular  substance  should  wear  a  mask  in  order  to  protect  him- 
self against  poisoning.  Vaughan  reports  that,  despite  this  precaution,  several  workers 
have  been  acutely  poisoned,  especially  with  the  typhoid  bacillus.  Of  course,  there 
is  no  danger  of  infection,  as  the  bacteria  are  killed  during  the  treatment.  If  the 
finely  ground  cellular  substance,  in  the  form  of  an  impalpable  powder,  is  kept  in  wide- 
mouthed  bottles  in  a  dark  place,  it  will  retain  its  toxicity  for  years.  This  powder 
constitutes  the  bacterial  protein  substance,  which  may  be  split  up  by  various  means. 
Vaughan  found  digestion  with  2  per  cent,  caustic  soda  in  absolute  alcohol  especially 
satisfactory  for  extracting  the  poisonous  group  from  bacterial  or  any  other  protein. 

A  weighed  portion  of  the  protein,  prepared  as  above,  is  placed  in  a  flask,  cov- 
ered with  from  fifteen  to  twenty-five  times  its  weight  of  absolute  alcohol  in  which 
2  per  cent,  of  sodium  hydroxid  has  been  dissolved.  The  flask,  fitted  with  a  reflux 
condenser,  is  heated  on  the  water-bath  for  one  hour,  where  it  is  allowed  to  cool  and 
the  insoluble  portion  collected  on  a  filter.  After  thorough  draining  the  insoluble 
part  is  returned  to  the  flask  and  the  extraction  repeated.  It  has  been  found  that 
three  extractions  are  necessary  in  order  to  split  off  all  the  poisonous  group.  The 
temperature  of  these  extractions  is  78°  C.,  the  temperature  of  boiling  absolute  alco- 
hol. By  this  method  the  protein  is  split  into  two  portions,  one  of  which  is  soluble  in 
absolute  alcohol  and  is  poisonous,  while  the  other  is  insoluble  in  absolute  alcohol  and 
is  not  poisonous  (Vaughan). 

Nature  of  Bacterial  Proteins. — Vaughan  and  his  coworkers  regard 
bacteria  as  essentially  parti culate,  specific  proteins.  He  has  not  been 
able  to  demonstrate  the  presence  of  cellulose  and  carbohydrates;  fats 


BACTERIAL    PROTEINS  129 

and  waves  that  may  be  present  are  somewhat  secondary  and  less  essen- 
tial constituents  or  stored  food  material.  The  sum  total  of  the  work  of 
these  observers  would  indicate  that  the  greater  part  of  bacteria  are  made 
up  of  true  proteins,  especially  nucleoproteins  or  glyconucleoproteins, 
and  although  they  may  be  simple  in  structure,  they  are  chemically 
complex — quite  as  much  so  as  many  of  the  tissues  of  the  higher  plants 
and  animals. 

When  bacterial  cellular  substances  are  split  up  with  mineral  acids 
or  alkalis  they  yield  ammonia,  mono-amino-  and  diamino-nitrogen,  one 
or  more  carbohydrate  groups,  and  humin  substances.  These  protein 
substances  are  the  same  as  those  obtained  by  the  hydrolysis  of  vegetable 
and  animal  proteins. 

By  digestion  with  dilute  acids  or  alkalis,  especially  the  latter,  in  the 
form  of  a  2  per  cent,  solution  of  sodium  hydroxid  in  absolute  alcohol,  a 
soluble  split  product  is  obtained  that  resembles  in  some  respects  the 
protamins,  although  they  do  not  all  give  a  satisfactory  biuret  reaction. 
This  product  is  highly  toxic,  but  shows  no  specificity  in  its  action,  being 
the  same  whether  derived  from  pathogenic  or  from  non-pathogenic  bac- 
teria, or  from  egg  albumin  or  other  protein  substance.  All  that  is  defi- 
nitely known  regarding  it  is  that  it  is  toxic,  protein  in  nature,  but 
simpler  in  structure  than  the  complex  proteins  of  the  bacterial  cells 
themselves. 

This  soluble  toxic  portion  as  obtained  in  vitro  is  regarded  by  Vaughan 
as  the  main  factor  in  the  production  of  the  general  symptoms  of  infection, 
the  special  and  distinctive  lesions  being  due  to  the  location  of  the  in- 
fection. During  the  infective  process  the  body-cells  produce  an  anti- 
ferment  which,  when  it  reaches  a  certain  concentration  or  power,  begins 
to  split  the  protein  of  the  microorganism  and  new  bacterial  tissue,  with 
the  liberation  of  this  toxic  moiety,  in  a  manner  similar  to  the.  splitting 
observed  in  vitro  by  dilute  alkalis  or  acids. 

The  insoluble  and  non-poisonous  portion  of  the  cellular  proteins 
shows  most  of  the  color  reactions  for  proteins,  and  contains  all  the  car- 
bohydrate of  the  unsplit  molecule  and  most  of  the  phosphorus. 

Action  of  Bacterial  Proteins. — The  effects  produced  by  bacterial 
proteins  are  not  specific;  the  protein  substance  of  non-pathogenic 
bacteria  and,  indeed,  many  proteins  derived  from  vegetable  and  animal 
sources,  have  equally  marked  pyogenic  properties.  All  foreign  proteins 
introduced  into  the  circulation  of  animals  are  more  or  less  toxic,  and  the 
toxic  effects  of  all  bacterial  proteins  are,  hi  general,  quite  similar  and 
non-specific. 


130  INFECTION 

Bacterial  protein  substances  may  be  responsible  for  certain  minor 
anaphylactic  reactions,  as  has  been  observed  occasionally  in  the  ad- 
ministration of  ordinary  bacterial  vaccines.  They  may  bear  an  im- 
portant relation  to  the  development  of  the  state  of  hypersensitiveness 
of  a  tuberculous  person  in  the  course  of  a  series  of  tuberculin  injections. 

Theory  of  Vaughan. — According  to  Vaughan  and  his  coworkers,  all 
true  proteins  contain  a  common  and  non-specific  poisonous  group. 
This  group  may  be  regarded  as  the  central  or  key-stone  portion  of  every 
protein  molecule,  with  secondary  and  possibly  tertiary  subgroups,  in 
which  the  specific  property  of  different  proteins  is  inherent.  When  the 
main  or  primary  group  is  detached  from  its  subsidiary  group,  it  mani- 
fests its  poisonous  action  by  the  avidity  with  which  it  attacks  the  second- 
ary group  of  other  proteins.  These  are  detached  from  their  normal 
positions,  and  consequently  deprive  the  living  protein  of  its  power  of 
functionating  normally.  When  proteins  are  split,  the  chemical  nucleus 
or  non-specific  toxic  portion  is  more  or  less  completely  set  free,  and  its 
toxicity  varies  according  to  the  thoroughness  with  which  the  secondary 
groups  have  been  removed. 

The  pathogenicity  of  a  bacterium  is  determined  not  by  its  capability 
of  forming  a  poison,  but  by  the  ability  it  possesses  to  grow  and  multiply 
in  the  animal  body.  When,  during  an  infection,  a  pathogenic  micro- 
organism reaches  the  deeper  tissues,  it  is  not  immediately  killed  by  the 
defensive  ferments  of  the  host,  but  continues  to  grow  and  multiply, 
throwing  out  a  ferment  that  feeds  upon  the  native  proteins  of  the  body- 
cells,  tearing  them  down  and  building  up  a  specific  bacterial  protein 
that  may  select  a  certain  point  of  predilection  in  which  it  is  most  prone 
to  accumulate.  Thus  the  typhoid  bacillus  accumulates  in  the  adenoid 
tissue  of  Peyer's  patches  on  the  intestine,  the  spleen,  and  the  mesenteric 
glands;  the  pneumococcus  tends  to  lodge  in  the  lungs;  the  smallpox 
virus  selects  the  skin,  etc. 

The  bacterial  toxins  and  viruses,  as,  e.  g.,  diphtheria  toxin  and  the 
virus  of  smallpox,  are  regarded  as  ferments  of  protein  nature,  capable 
of  attacking  native  body  protein  and  building  up  a  specific  foreign  pro- 
tein. This  foreign  bacterial  protein  is  formed  during  the  period  of 
incubation  of  disease  when  there  is  no  effective  resistance  on  the  part 
of  the  body-cells  to  its  growth  and  multiplication.  During  this  time 
the  infected  person  is  not  ill,  so  that  the  foreign  protein  in  itself  cannot 
be  toxic,  and  the  body-cells  are  busy  preparing  and  elaborating  a  new 
and  specific  ferment  that  will  digest  and  destroy  the  foreign  protein. 
When  this  new  ferment  becomes  active,  the  first  symptoms  of  disease 


PTOMAINS  131 

appear,  and  the  active  stage  of  the  disease  marks  the  period  over  which 
the  parenteral  digestion  of  the  foreign  protein  extends.  These  specific 
ferments  split  up  the  foreign  protein  and  liberate  the  toxic  portion  or  the 
protein  poison;  this  poison  is  not  a  toxin  and  is  not  specific,  but  occurs 
commonly  in  all  proteins. 

The  characteristic  symptoms  and  lesions  caused  by  the  various 
infectious  processes  are  determined  largely  by  the  location  of  the  foreign 
protein.  The  poison  elaborated  is  the  same  in  all  infectious  diseases, 
and  it  is  the  location  of  the  infection,  rather  than  the  exact  nature  of  the 
infecting  agent,  which  gives  rise  to  the  more  or  less  characteristic  symp- 
toms and  lesions  of  the  several  infectious  diseases. 

Death  may  be  produced  by  the  too  rapid  breaking-up  of  the  foreign 
protein,  and  the  consequent  liberation  of  a  fatal  dose  of  the  protein 
poison,  or  it  may  result  from  a  lesion  induced  by  the  products  of  this 
disruption,  such  as  perforation ,  of  the  intestine  and  hemorrhage  in 
typhoid  fever,  or  it  may  follow  from  chronic  intoxication  and  consequent 
exhaustion.  If  recovery  takes  place,  the  individual  enjoys  an  immunity 
of  variable  duration,  owing  to  the  presence  of  specific  ferments  capable 
of  destroying  the  particular  substrata  if  infection  should  occur. 

It  is  this  power  of  body-cells,  when  permeated  by  a  foreign  protein, 
to  elaborate  a  specific  antiferment  by  which  the  protein  is  destroyed, 
that,  in  the  opinion  of  Vaughan,  forms  the  basis  of  a  correct  understand- 
ing of  infection  and  immunity.  * 


PTOMAINS 

It  was  at  one  time  believed  that  the  symptoms  of  many  diseases 
were  due  to  the  absorption  of  soluble  basic  nitrogenous  substances  pro- 
duced by  bacterial  action  upon  various  albumens,  these  toxic,  alkaloid- 
like  substances  being  known  as  ptomains.  It  was  soon  found,  however, 
that  the  ptomains  produced  by  pathogenic  bacteria  were  insufficient  of 
themselves  to  cause  the  symptoms  and  lesions  characteristic  of  the  re- 
spective microorganisms;  that  they  were  in  general  less  toxic  than  the 
cultures  themselves;  that  the  majority  of  ptomains  are  not  very  poison- 
ous; and  that  they  are  not  specific,  since  equally  potent  ptomains  are 
produced  by  non-pathogenic  bacteria.  This  lack  of  specificity  is  in 
sharp  contrast  to  the  toxins.  No  matter  upon  what  medium  a  true 
toxin  producer  is  grown,  the  toxin  is  qualitatively  the  same,  whereas  the 
nature  and  toxicity  of  ptomains  depend  upon  the  microorganism,  the 
culture-medium  used,  the  duration  of  growth,  and  the  quantity  of  oxygen 


132  INFECTION 

furnished.  The  same  microorganism,  when  grown  on  different  media 
or  under,  different  conditions,  may  produce  totally  different  ptomains. 

Ptomains  may,  however,  produce  disease,  and  even  death,  when  they 
are  ingested  with  food  that  has  undergone  bacterial  decomposition.  In 
most  instances  of  meat-poisoning,  however,  which  are  frequently  as- 
cribed to  the  presence  of  ptomains,  a  specific  microorganism,  the 
Bacillus  botulinus,  or  a  member  of  the  Bacillus  enteritidis  group  of 
Gartner,  is  usually  responsible.  The  commonest  sources  of  ptomain 
poisoning  are  improperly  preserved  meats,  fish,  sausages,  cheese,  ice- 
cream, and  milk.  This  subject  received  full  consideration  in  Vaughan 
and  Novy's  "Cellular  Toxins." 

A  number  of  ptomains  are  known,  and  of  some  the  exact  chemical 
constitution  has  been  established.  Brieger  has  separated  one  from 
decomposing  flesh  and  cholera  cultures,  called  cadaverin,  which  Laden- 
burg  has  shown  to  be  pentamethylendiamin,  and  prepared  synthetically. 
Muscarin,  isolated  by  Schmiedberg  and  Brieger,  and  tyrotoxicon,  iso- 
lated by  Vaughan,  are  also  well  known. 

In  the  isolation  of  bacterial  ptomains  Brieger's  method  is  generally 
employed,  which  consists  of  acidulating  large  amounts  of  culture  with 
hydrochloric  acid,  boiling,  filtering,  evaporating  the  filtrate  to  a  syrupy 
consistency,  dissolving  in  96  per  cent,  alcohol,  and  precipitating  and 
purifying  by  means  of  an  alcoholic  solution  of  mercuric  chlorid. 

Besides  occurring  in  food-poisoning,  ptomains  may  be  formed  as  the 
result  of  putrefactive  processes  going  on  in  abscesses,  gangrenous  areas, 
and  within  the  gastro-intestinal  canal,  and  enough  of  these  may  be 
absorbed  to  produce  symptoms  of  intoxication.  Under  these  condi- 
tions it  is  possible  for  bacteria  to  produce  ptomains  that  may  be  absorbed 
and  produce  symptoms  of  intoxication  without  the  bacteria  themselves 
actually  gaining  entrance  to  the  tissues,  and  therefore  not  constituting, 
according  to  our  definition,  a  true  infection.  Pernicious  anemia, 
chlorosis,  and  allied  conditions  have  been  ascribed  to  the  absorption  of 
such  ptomains  from  the  intestinal  canal.  Obviously  it  is  difficult  or  im- 
possible to  always  differentiate  between  bacterial  toxins  and  bacterial 
ptomains,  or  the  products  of  protein  decomposition  dependent  upon 
bacterial  activity,  and  we  can  but  admit  the  possibility  of  the  produc- 
tion and  absorption  of  both  bacterial  toxins  and  ptomains  under  certain 
pathologic  conditions.  Most  ptomains  probably  are  produced  as  the  re- 
sult of  decomposition  of  the  dead  protein  medium  upon  which  the  bacteria 
grow,  and  to  a  lesser  extent  by  the  destruction  of  the  bacterial  cells  them- 


MECHANICAL  ACTION   OF   BACTERIA  133 

selves.     It  is  extremely  doubtful  if  ptomains  are  produced  in  important 
quantities  by  pathogenic  bacteria  infecting  living  tissues. 


MECHANICAL  ACTION  OF  BACTERIA 

In  former  years  the  theory  as  to  the  influence  of  mechanical  blocking 
of  vessels  with  masses  of  bacteria  was  regarded  with  much  favor  in  the 
etiology  of  certain  infections,  particularly  anthrax.  At  the  present 
time  this  factor  has  not  the  same  importance,  for  while  it  is  true  that 
bacterial  emboli  may  occasion  harm  by  blocking  important  vessels, 
further  researches  have  shown  that  it  is  doubtful  if  any  pathogenic 
microorganisms  are  totally  devoid  of  toxic  action,  and  that  their  toxins 
are  largely  responsible  for  the  tissue  changes  and  symptoms  of  infections. 

It  can  readily  be  understood  that  emboli  of  microorganisms  may 
produce  metastatic  lesions;  thus  when  staphylococci  are  injected  into 
the  ear  vein  of  a  rabbit  they  produce  abscesses  in  the  kidney  and  heart, 
and  masses  of  bacteria  from  an  ulcerative  endocarditis,  when  carried  to 
different  portions  of  the  body,  will  cause  abscess  formation;  but  the 
question  in  hand,  however,  deals  with  the  effects  of  mechanical  blocking 
itself. 

Investigations  with  anthrax  bacilli  have  shown  that  they  are  re- 
markably free  from  soluble  toxins  and  endotoxins,  although  the  local 
lesion  develops  so  rapidly  and  becomes  so  quickly  necrotic  as  to  suggest 
very  strongly  the  action  of  some  local  toxic  substance.  Cases  of  human 
anthrax  seldom  prove  fatal  if  the  lesion  is  removed  and  the  blood-stream 
remains  free  from  the  bacilli.  Vaughan  has  shown  that  anthrax  protein 
possesses  toxic  qualities,  and  since  the  majority  of  fatal  cases  of  anthrax 
show  enormous  numbers  of  bacilli  in  the  blood-stream  and  internal 
organs,  it  may  be  that  this  bacteremia  produces  an  accumulation  of 
toxins  which  is  greatly  augmented  when  the  body-cells  of  the  host  have 
produced  an  antiferment  that  splits  up  the  protein  of  the  bacilli,  the 
combined  toxic  substances  being  responsible  for  the  severe  symptoms 
and  death. 

With  protozoan  disease,  the  possibility  of  serious  symptoms  follow- 
ing blocking  of  vessels  is  far  greater,  and,  indeed,  the  cerebral  symp- 
toms of  malignant  malaria  and  sleeping  sickness  may  be  due  in  part 
to  the  blocking  of  small,  but  physiologically  important,  vessels  with 
masses  of  plasmodia  and  Trypanosoma  gambiensi,  together  with  the  ab- 
sorption of  toxic  agents  and  the  products  of  disintegration.  Thus  Bass, 
who  has  successfully  cultivated  the  malarial  plasmodium  outside  of  the 


134  INFECTION 

body,  believes  that  the  parasites,  after  attaining  sufficient  size,  lodge  in 
the  capillaries  of  the  body,  especially  where  the  blood-current  is  weakest, 
and  where  slight  obstruction  occurs  as  the  result  of  the  protruding  in- 
ward of  nuclei  of  the  endothelial  cells.  Here  they  remain  and  develop 
until  segmentation  takes  place.  In  the  meantime  other  red  corpuscles 
are  forced  against  them,  and  if  the  opening  in  the  infected  cell  is  in  a 
favorable  location,  one  or  more  merozoites  pass  directly  into  another 
cell;  if  it  is  not,  the  merozoites  are  discharged  into  the  blood-stream  and 
are  speedily  killed. 

INFECTION  WITH  ANIMAL  PARASITES 

Infection  with  animal  parasites  is  similar  in  many  respects  to  in- 
fection with  bacteria.  Owing  to  the  difficulty  of  isolating  and  culti- 
vating these  parasites  in  vitro,  our  knowledge  of  their  toxic  properties 
is  somewhat  meager.  Most  attention  has  been  given  to  a  study  of 
their  life  history  and  the  modes  of  transmission. 

Modes  of  Infection. — Primary  infection  with  animal  parasites  is 
often  facilitated  by,  or  in  some  instances  only  rendered  possible  through, 
the  intervention  of  special  carriers,  usually  various  species  of  the  Ar- 
thropoda.  Thus  we  now  know  that  malaria  is  transmitted  through  the 
bite  of  infected  mosquitos;  African  relapsing  fever  and  Texas  cattle 
fever,  through  the  bite  of  certain  infected  ticks;  trypanosomiasis, 
through  biting  flies.  The  ova  of  various  intestinal  parasites  may  re- 
quire residence  in  certain  of  the  lower  animals  before  they  can  infect 
man. 

Infection  may  occur  along  the  same  routes  as  bacterial  infection, 
and  is  governed  in  general  by  the  same  factors  of  local  selection,  tissue 
susceptibility,  etc.  Biting  insects  usually  deposit  the  parasite  directly 
in  the  subcutaneous  tissues  or  in  the  circulatory  fluids.  Abrasion  of 
the  epithelium  may  be  necessary  in  order  to  produce  infection  with 
Treponema  pallidum,  as  in  the  majority  of  the  bacterial  infections. 
The  ova  or  larva  of  other  parasites  may  be  swallowed  or  find  lodgment 
in  the  upper  or  lower  air-passages  or  accessory  sinuses. 

It  would  appear  that  our  natural  defenses  against  infection  with 
animal  parasites  are  much  weaker  than  those  against  bacteria;  this  is 
probably  due  to  the  greater  resistance  offered  by  animal  parasites  to 
such  physical  destructive  influences  of  the  host,  as  the  acidity  and  ger- 
micidal  activity  of  the  secretions,  temperature,  etc.,  as  well  as  to  a 
general  lack  of  natural  antibodies  in  the  body-fluids  of  the  host,  and 
inability  of  leukocytes  and  other  phagocytic  cells  to  deal  successfully 


INFECTION   WITH   ANIMAL   PARASITES  135 

with  the  invaders.  That  natural  immunity  against  infection  with 
certain  animal  parasites  may  exist  is  shown  by  the  prevalence  of  certain 
infections  among  man,  and  their  absence  among  lower  animals,  or  vice 
versa. 

The  aggressiveness  of  animal  parasites  is  in  general  probably  even 
greater  than  that  of  most  bacteria,  and  a  more  or  less  extensive  infection 
apparently  occurs  in  all  cases  in  which  the  parasite  had  made  successful 
invasion,  some  multiplying  in  the  blood-stream  (malaria,  relapsing 
fever,  trypanosomiasis,  Texas  fever,  filariasis),  others  in  the  lymph- 
stream  (filariasis),  and  others  in  the  tissues  (syphilis,  trichiniasis, 
amebiasis)  without  much  opposition  on  the  part  of  the  host.  Whether 
these  factors  are  due  to  the  aggressive  forces  of  the  parasites  which  neutra- 
lize the  defenses  of  the  host,  or  whether  they  are  due  to  the  hardiness  of 
the  parasites  and  a  lack  of  defense  on  the  part  of  the  host,  is  not  known, 
but  probably  the  latter  is  generally  the  case. 

As  with  bacteria,  animal  parasites  show  a  well-marked  selective 
affinity  for  certain  tissues,  as  the  malarial  plasmodium  for  red  blood- 
corpuscles,  trypanosomes  and  spirochetes  for  blood  plasma,  trichina 
for  voluntary  muscle,  various  parasites  for  the  intestinal  canal  and  even 
for  certain  portions  of  the  intestinal  tract,  others  for  the  lung,  etc. 

Production  of  Disease. — Comparatively  little  is  known  regarding 
the  formation  of  toxic  products  on  the  part  of  the  animal  parasite. 
Some,  as,  e.  g.,  the  Treponema  pallidum  and  spirochete  of  relapsing 
fever,  probably  cause  disease  largely  through  the  production  of  toxins, 
especially  of  the  intracellular  variety.  The  chill,  fever,  and  sweat  of 
malaria  suggest  the  liberation  of  toxic  products  coincident,  or  nearly  so, 
with  segmentation  and  rupture  of  the  plasmodium.  The  late  symptoms 
of  sleeping  sickness  and  the  whole  course  of  relapsing  fever  are  strikingly 
similar  to  the  bacterial  toxemias.  The  metabolic  products  of  all  animal 
parasites  are  probably  injurious  in  some  manner  and  to  some  degree. 
The  pathogenicity  of  others  is  due,  in  part  at  least,  to  mechanical 
blocking  of  vessels,  as  with  the  filaria,  trypanosomes,  and  malarial 
organisms ;  others  (hookworm)  abstract  blood  or  consume  food  material 
in  the  intestine,  as  the  intestinal  parasites;  and  others,  as  migrating 
foreign  particles  with  irritating  secretions,  produce  local  inflammatory 
changes. 

Nevertheless,  we  know  comparatively  little  of  the  offensive  factors, 
and  still  less  of  the  immunologic  defensive  factors,  operative  during  the 
course  of  infections  with  animal  parasites.  With  the  development  of  a 
technic  for  the  cultivation  of  animal  parasites  in  vitro  similar  to  that 


136  INFECTION 

devised  for  the  ameba,  certain  trypanosomes,  spirochetes,  and  malarial 
plasmodia,  we  will  be  enabled  to  study  the  products  of  their  growth  or 
of  disintegration,  and  the  immunologic  agencies  concerned  in  infection 
and  recovery;  this  offers  a  very  important  and  fruitful  field  for  research. 


THE  COURSE  OF  INFECTION 

In  conclusion,  we  may  briefly  consider  the  results  of  infection  or  the 
general  symptoms  following  bacterial  growth  and  the  manner  in  which 
these  are  produced. 

The  Stages  of  Infection. — Practically  all  infections  pass  through  the 
following  stages : 

1.  The  period  of  incubation,  which  begins  at  the  time  of  infection  and 
ends  with  the  development  of  the  earliest  general  symptoms,  during 
which  time  the  invading  bacteria  are  multiplying  in  the  tissues  of  the 
host.  During  this  stage  no  symptoms,  or  only  those  of  a  purely  local 
nature,  are  present.  This  period  varies  considerably  in  different  in- 
fections, and  to  a  lesser  extent  in  different  individuals  having  the  same 
infection.  Some  bacteria  may  be  so  virulent  as  to  overwhelm  the  body- 
cells,  thus  making  the  period  of  incubation  very  short  or  entirely  un- 
observable.  On  the  other  hand,  as,  e.  g.,  in  rabies,  the  period  may  be  of 
several  weeks'  and,  indeed,  of  several  months'  duration.  In  tuberculosis 
there  is  usually  a  primary  local  growth,  which  develops  so  gradually 
and  the  toxins  are  so  slowly  diffused  that  it  is  difficult  or,  indeed,  im- 
possible, to  estimate  the  length  of  the  period  of  incubation. 

According  to  Vaughan,  during  the  period  of  incubation  the  bacteria 
or  their  toxins  or  the  viruses  are  actively  engaged  in  changing  the  natural 
body  proteins  into  new  and  specific  bacterial  proteins,  and  since  this 
stage  is  constructive,  there  are  no  symptoms  and  the  host  is  not  ill. 
Even  with  the  experimental  administration  of  the  most  poisonous  of 
toxins  a  definite  period  of  incubation  is  usually  to  be  observed,  which 
cannot  be  reduced  below  a  certain  minimum,  independent  of  the  size 
of  the  dose  injected;  in  general,  however,  a  large  dose  of  bacteria  or  of 
toxin  is  likely  to  be  followed  by  a  shorter  period  of  incubation  than  if  a 
smaller  dose  were  administered. 

In  a  given  case  the  period  of  incubation  may  be  determined  by 
several  factors: 

(a)  The  number  of  bacteria  gaining  entrance,  and  especially  their 
toxicity  and  aggressiveness.  The  primary  factors  are  the  degree  of 
toxicity  and  the  amount  of  toxic  substances  produced  and  absorbed. 


THE   COURSE    OF   INFECTION  137 

(6)  Upon  the  site  of  infection.  Thus  the  introduction  of  rabies 
virus  or  of  tetanus  bacilli  into  the  tissues  of  the  face  or  into  a  deep  wound 
is  likely  to  be  followed  by  a  shorter  period  of  incubation  than  when 
these  are  introduced  into  the  foot  or  in  superficial  wounds. 

(c)  Upon  the  degree  of  resistance  offered  by  the  host.     For  instance, 
one  individual  may  contain  more  antitoxin  or  bacteriolysin  for  a  certain 
bacterium  than  another,  and  consequently  a  longer  period  of  incubation 
is  required,  during  which  these  substances  are  neutralized  and  an  excess 
of  toxic  bacterial  substance  is  produced.     In  fact,  these  may  offer  such 
resistance  to  the  bacterium  that  the  process  of  infection  is  inhibited,  or 
but  slight  and  evanescent  disturbances  appear. 

(d)  Upon  the  general  susceptibility  of  the  host  and  the  route  of  in- 
vasion. 

2.  The  period  of  prodromal  symptoms,  characterized  by  systemic 
listurbances  of  a  relatively  mild  type,  due  to  diffusion  of  the  bacteria 

and  their  products  into  the  general  circulation  and  their  wide-spread 
effect  upon  the  body-cells  in  general.  If  the  bacteria  select  a  special 
tissue  or  organ  for  attack,  as  the  typhoid  bacillus  for  lymphoid  tissue, 
and  pneumococci  for  the  lungs,  definite  symptoms  develop  later,  their 
nature  depending  on  the  special  tissue  or  organ  involved.  The  pro- 
dromata,  however,  are  more  marked,  and  indicate  a  wide-spread  but 
mild  action  upon  the  body-cells  in  general.  Vaughan  believes  that  these 
symptoms  mark  the  time  when  sufficient  proteolytic  ferments  have  been 
generated  by  the  body-cells  against  the  new  bacterial  protein  of  the 
invading  bacteria  to  attack  the.  latter,  splitting  the  molecule  and  liberat- 
ing a  toxic  moiety  responsible  for  the  general  symptoms  of  intoxication. 

3.  The  period  of  fastigium,  or  of  high  fever,  during  which  the  disease 
is  at  the  height  of  its  severity.     Special  and  distinctive  symptoms  and 
lesions,  according  to  the  organ  or  organs  especially  involved,  are  present; 
the  struggle  between  the  offensive  and  defensive  forces  of  parasite  and 
host  is  at  its  height,  with  remissions  or  exacerbations  dependent  upon 
the  supremacy  of  any  one  of  these,  and  the  general  stability  of  the  body- 
cells  in  withstanding  the  wear  and  tear.     During  this  time  the  protect- 
ive proteolytic  ferments  of  Vaughan  are  most  active  in  disrupting  the 
newly  formed  bacterial  protein,  with  the  liberation  of  the  toxic  portion. 
This  process  may  be  so  active  as  to  overwhelm  the  host  with  the  toxic 
split  product,  or  lead  to  grave  secondary  lesions,  such  as  extensive  necro- 
sis, perforation  of  a  viscus,  or  hemorrhage. 

4.  The  period  of  decline,  during  which  the  patient  is  gradually  over- 
coming the  infection,  and  amelioration  of  the  symptoms  takes  place. 


138  INFECTION 

5.  The  period  of  convalescence  is  now  ushered  in,  during  which  the 
host  gradually  overcomes  the  effects  of  disease  and  returns  to  health. 

During  this  entire  time  the  emaciation  and  tissue  exhaustion  leave 
the  patient  quite  weak,  and  undue  exertion,  errors  in  diet,  or  reinfection 
may  lead  to  a  relapse,  or  a  reactivation  of  the  disease.  Certain  sequelae 
or  morbid  conditions  may  follow  a  disease,  and  are  due  to  the  same  orig- 
inal cause;  e.  g.,  in  typhoid  fever  the  development  of  cholecystitis;  at 
any  time  during  the  disease  complications,  or  morbid  conditions  due 
to  some  other  microparasite,  as  the  development  of  pneumonia  during 
the  course  of  typhoid  fever,  may  seriously  jeopardize  the  life  of  the 
patient. 

Grades  of  Infection. — According  to  the  manner  in  which  a  parasite 
and  its  products  act  upon  the  cell  of  a  host  and  the  power  of  the  host  to 
neutralize  or  overcome  these  the  following  various  grades  and  types  of 
infection  are  encountered : 

(a)  Malignant  or  fulminating  infection,  during  which  there  is  no 
fever,  but,  on  the  contrary,  a  subnormal  temperature,  with  rapid  prostra- 
tion of  the  patient  and  death  within  a  brief  period.     The  cells  of  the  body 
are  overwhelmed  and  paralyzed  by  the  toxic  substances;    metabolism 
is  arrested,  and  the  heat  centers  are  exhausted  with  the  fall  of  the  tem- 
perature, an  indication  of  the  intense  and  overwhelming  intoxication. 

(b)  Acute  infection,  which  is  the  ordinary  type  of  an  infectious  disease 
as  previously  described,  and  having  a  definite  incubation  period,  prodro- 
mal symptoms,  fastigium,  defervescence,  and  convalescence. 

(c)  Chronic  infection,  or  a  prolonged  process  characterized  by  in- 
sidious onset  and  symptoms  of  relatively  mild  or  moderate  severity, 
and  terminating  either  in  death,  after  months  or  years,  or  in  gradual 
recovery.     A  chronic  infection  may  be  remittent,  as  may  be  observed 
in  the  rheumatic  group  of  disorders;    during  the  remission  with  defer- 
vescence the  infecting  bacterium  is  not  totally  destroyed,  -  and  subse- 
quently lights  up,  producing  an  acute  exacerbation  of  the  disease. 

In  chronic  infections  it  would  appear  that  the  parasites  develop  and 
produce  their  toxins  slowly,  or  that  these  are  slowly  and  imperfectly 
absorbed  on  account  of  the  sluggish  local  circulation  and  the  presence 
of  scar  tissue.  The  body-cells  become  accustomed,  as  it  were,  to  these 
toxic  products,  and  produce  only  sufficient  antibodies  to  effect  their  imme- 
diate neutralization.  The  bacteria  themselves  become  distinctly  resistant 
to  the  action  of  the  tissues  and  the  defensive  forces,  and  there  is  neither 
the  same  degree  of  intoxication  nor  reaction  as  are  seen  in  acute  infec- 
tions. Gradually,  however,  the  body-cells  become  exhausted,  and 


THE    COURSE    OF    INFECTION  139 

unless  the  cells  are  aroused  and  stimulated,  by  judicious  administration 
of  bacterial  vaccines,  to  produce  an  oversupply  of  antibodies,  the  host 
shows  progressive  emaciation  and  weakness. 

The  Systemic  Reaction  to  Infection. — It  is  not  within  the  scope  of 
this  book  to  discuss  the  various  symptoms  of  infection,  and  we  will 
limit  ourselves  to  a  brief  discussion  of  the  most  important,  namely,  the 
febrile  reaction. 

According  to  Vaughan,  the  fever  of  infection  is  due  mainly  to  the 
toxic  split  protein  resulting  from  the  action  of  the  protective  proteolytic 
ferments  upon  the  new  bacterial  protein.  This  observer  and  his  asso- 
ciates were  able,  by  the  injection  of  multiple  doses  of  protein  derived 
not  only  from  the  typhoid  bacillus  but  from  various  vegetable  and 
animal  proteins,  to  reproduce  experimentally  in  rabbits  a  febrile  reaction 
known  as  protein  fever,  and  which  is  not  unlike  typhoid  fever.  This 
induced  fever  may  continue  for  weeks,  and  is  accompanied  by  increased 
nitrogen  elimination  and  gradual  wasting;  it  is  followed  by  immunity, 
and  the  serum  of  immunized  animals  digests  the  homologous  protein 
in  vitro.  As  has  repeatedly  been  stated,  Vaughan  regards  the  split  toxic 
product  as  the  cause  of  the  general  symptoms  of  infection,  the  special 
and  characteristic  symptoms  and  lesions  of  the  different  diseases  de- 
pending upon  the  site  where  the  bacterial  proteins  have  been  deposited, 
and  where  they  are,,  in  large  part  at  least,  digested. 

In  addition  to  this  toxic  action  of  split  protein,  fever  may  be  due — 
(a)  to  the  unusual  activity  of  the  cells  supplying  the  proteolytic  enzymes; 
and  (6)  to  the  cleavage  of  the  foreign  bacterial  protein  by  these  ferments. 

The  fever  of  infection,  therefore,  is  caused  by  the  toxic  action  of 
pathogenic  parasites,  both  bacterial  and  animal  forms,  upon  the  body- 
cells  and  heat-regulating  centers.  It  must  be  regarded  by  itself  as  a 
beneficent  phenomenon,  inasmuch  as  it  marks  a  reaction  of  the  body- 
cells  to  toxic  agents,  for  the  purpose  of  neutralizing  these  and,  by  the 
development  of  antibodies,  ridding  the  body  of  foreign  substances. 


PART  III 

CHAPTER  VIII 

IMMUNITY,— THEORIES  OF  IMMUNITY 

IN  the  preceding  chapter  on  the  mechanism  of  infection  and  the  pro- 
duction of  an  infectious  disease  the  statement  was  frequently  made  that 
the  microparasites  of  disease  are  required  to  overcome  the  defensive 
forces  of  a  host  which  are  ever  on  guard  to  protect  the  organism  against 
parasitic  invasion  and  infection.  Certain  of  these  defenses  are  natural 
to  the  host,  and  in  a  great  majority  of  instances  suffice  to  protect  the 
body  against  invasion  and  infection  with  bacteria,  animal  parasites,  and 
various  inanimate  and  injurious  substances.  When,  however,  these 
natural  defenses  are  broken  down  and  infection  has  occurred,  the  body- 
cells  are  not  usually  rendered  powerless  and  completely  overcome,  for 
the  products  of  infection  serve  as  a  stimulus  to  the  body-cells,  calling 
forth  renewed  cellular  activity  and  the  production  of  various  specific 
defensive  weapons,  termed  antibodies,  which  maintain  an  incessant 
struggle  against  the  invading  pathogenic  agents  in  an  effort  to  rid  the 
body  of  them  and  to  neutralize  their  products. 

Just  as  microparasites  have  various  offensive  weapons,  consisting 
chiefly  of  their  toxins,  so,  in  like  manner,  the  defensive  forces  of  the  host 
are  numerous  and  even  more  complex.  If  the  toxin  of  a  microorganism 
is  its  chief  pathogenic  weapon,  as,  e.  g.,  the  soluble  and  extracellular 
toxin  of  the  diphtheria  or  the  tetanus  bacillus,  then  the  body-cells  pro- 
duce an  antitoxin  as  their  chief  defensive  force.  If  the  offensive  weapon 
is  largely  in  the  nature  of  an  endotoxin,  as,  for  example,  the  endotoxins 
of  the  typhoid  or  the  cholera  bacillus,  then  a  chief  antibody  is  in  the 
nature  of  a  bacteriolysin,  which  endeavors  to  dissolve  the  bacillus  in  an 
effort,  as  it  were,  to  attack  the  enemy  in  his  stronghold.  In  other*  in- 
fections, especially  those  due  to  the  pyogenic  cocci,  certain  of  the  body- 
cells,  and  chiefly  the  polynuclear  leukocytes,  are  observed  in  the  tissues 
to  have  engulfed  the  invaders  bodily  (phagocytes)  in  an  endeavor  to 
digest  them  and  neutralize  their  products.  In  addition  to  these  chief 
antibodies,  there  are  others  that  appear  to  aid  them  in  their  work. 

That  one  attack  of  many  of  the  infectious  diseases  may  protect  the 
individual  against  subsequent  attacks,  or  at  least  render  subsequent 
attacks  mild  and  harmless,  is  well  known.  In  India  and  the  East  for 

140 


THEORIES    OF   IMMUNITY  141 

centuries  practical  advantage  has  been  taken  of  this  observation  in  the 
management  of  smallpox.  In  order  to  protect  persons  against  a  severe 
attack  of  variola  they  were  deliberately  brought  in  contact  with  a 
person  suffering  with  a  particularly  mild  form,  in  the  hope  that,  by 
inducing  a  mild  attack  of  short  duration,  they  would  thus  obtain  pro- 
tection against  the  severe,  disfiguring,  and  fatal  form  of  the  disease. 

The  practice,  however,  was  not  without  danger  to  the  individual  and 
to  the  public  at  large,  as  the  induced  disease  would  at  times  become 
malignant,  and  constitute  a  focus  of  infection  for  an  entire  community, 
When  Edward  Jenner  discovered  that  inoculation  with  cowpox  virus 
could  not  produce  smallpox,  but  would,  nevertheless,  stimulate  the  pro- 
duction of  specific  antibodies  and  confer  immunity  against  it,  an  enor- 
mous forward  stride  was  taken  that  has  since  proved  a  priceless  boon  in 
helping  to  rid  the  world  of  the  dreadful  scourge  of  smallpox. 

The  object  of  all  these  procedures  has  been  to  secure  a  resistance  or 
immunity  to  smallpox,  either  by  inducing  a  mild  form  of  the  disease  or 
by  protecting  the  individual  by  means  of  inoculation  with  a  virus  that 
has  been  so  changed  in  its  passage  through  a  cow  as  to  render  it  unable 
to  produce  smallpox,  but  yet  is  capable  of  stimulating  the  body-cells  to 
produce  antibodies  that  will  neutralize  the  effect  of  the  true  virus.  This 
induced  resistance  to  a  given  infection  constitutes  immunity  or  resist- 
ance, and  since  the  body  was  purposely  inoculated  and  the  body-cells 
rendered  active  in  producing  the  antibodies,  this  form  of  resistance  is 
known  as  active  acquired  immunity. 

Many  persons  recover  from  an  infection  that  may  have  been  unusu- 
ally severe  not  because  the  infecting  agent  became  exhausted  or  died  for 
want  of  pabulum,  but  because  it  had  been  gradually  worsted  in  the 
battle  with  the  defensive  forces  of  the  host.  In  many  such  instances 
the  host  is  now  immune  to  this  infection  for  a  longer  or  shorter  time, 
because  the  body-cells  have  been  so  profoundly  impressed  that  they  con- 
tinue generating  defensive  weapons  or  antibodies  for  some  time  after 
the  last  vestige  of  the  infecting  agent  has  disappeared.  Or,  on  the  other 
hand,  the  quantity  of  antibodies  may  be  so  great  that  they  may  persist 
for  varying  periods  of  time,  even  for  the  remainder  of  life,  ever  on  guard, 
and  ready  to  overwhelm  their  specific  enemy  should  it  ever  again  gain 
access  to  the  tissues. 

Here,  then,  arises  the  question  concerning  the  mechanism  of  re- 
covery from  an  infection,  and  since  this  is  so  intimately  concerned  with 
the  general  subject  of  resistance  to  disease,  it  is  considered  under  the 
general  head  of  immunity. 


142  IMMUNITY. — THEORIES    OF   IMMUNITY 

Even  superficial  observation  shows  that  not  all  persons  are  equally 
susceptible  to  a  given  disease,  and  during  the  course  of  epidemics  it  will 
be  seen  that  some  individuals,  although  freely  exposed,  escape  infection. 
Certain  species  of  animals  may  likewise  display  a  uniform  resistance  to 
an  infection  that  will  readily  enough  attack  another  species  of  the  same 
general  family.  It  has  been  demonstrated  experimentally  that  a  certain 
pathogenic  bacterium  will  produce  a  severe  infection  in  one  species  of 
animals  and  not  in  another.  It  may  frequently  be  noticed  that  even 
though  an  infection  occurs,  it  is  readily  overcome  by  the  natural  resources 
of  the  host,  the  latter  escaping  with  slight  or  no  symptoms  of  disease. 
In  other  words,  certain  persons  and  animals  apparently  possess  a  natural 
resistance  or  immunity  to  disease,  which  may  be  general,  non-specific, 
or  due  to  specific  antibodies,  this  type  of  immunity  being  frequently 
relative  and  seldom  absolute. 

Definition. — Immunity,  therefore,  in  a  broad  sense,  is  the  effective 
resistance  of  the  organism  against  any  deleterious  influence;  in  the  usual 
and  more  restricted  meaning  the  term  is  applied  to  resistance  against  in- 
fection with  vegetable  and  animal  parasites  and  their  products,  which  are 
pathogenic  for  other  animals  of  the  same  or  of  different  species. 

It  should  be  remembered  that  immunity  means  not  only  the  ability 
to  resist  an  infection  or  successful  invasion  of  the  tissues  by  micropara- 
sites,  but  also  the  continual  resistance  offered  as  long  as  the  infection 
lasts;  that  is,  immunity  implies  not  only  resistance  to  the  onset  of  in- 
fection, but  also  to  the  course  and  progress  of  the  resulting  infectious 
disease.  The  science  of  immunity  has,  therefore,  for  its  object  the  study 
of  the  mechanism  of  resistance  to  and  recovery  from  an  infection. 


HISTORIC 

The  development  of  the  science  of  immunity  forms  one  of  the  most 
interesting  chapters  in  the  history  of  medicine.  Even  in  ancient  history 
we  can  trace  the  conception  of  our  modern  ideas  on  immunization. 

Hippocrates  taught  that  the  factor  that  causes  a  disease  is  also 
capable  of  curing  it — practically  the  same  theory  as  the  more  modern 
homeopathic  doctrine  of  ((similia  similibus  curantur."  Pliny  the  Elder 
recommended  the  livers  of  mad  dogs  as  a  cure  for  hydrophobia,  thus 
coming  very  near  to  the  basis  of  the  Pasteur  discovery.  As  was  pointed 
out  by  Elizabeth  Fraser,  the  same  idea  is  expounded  in  the  mythologic 
tale  of  Telephus,  who  cured  his  wound  by  applying  rust  from  the  sword 
which  inflicted  it,  and  in  the  story  of  Mithridates,  King  of  Pont  us  (B. 


HISTORIC 


143 


C.  120),  who  immunized  himself  against  poisons  by  drinking  the  blood 
of  ducks  that  had  been  treated  with  the  corresponding  toxic  substances. 

Immunization  against  various  venoms  has  been  practised  by  many 
of  the  savage  tribes  of  Africa  since  earliest  times.  Mention  has  previ- 
ously been  made  of  the  method  of  preventive  inoculation  against  small- 
pox practised  in  Asia  and  other  Oriental  countries  for  several  centuries 
by  exposing  the  subjects  to  mild  cases  of  the  disease. 

A  very  definite  step  in  progress  must  ever  be  associated  with  the 
name  of  Edward  Jenner,  who  first  demonstrated  experimentally,  and 
in  a  scientific  manner,  that  cowpox  conveyed  to  man  protected  him 
against  smallpox.  Jenner  was  not  the  first  person  to  make  this  observa- 
tion, as  many  of  the  Gloucestershire  farmers  knew  that  cowpox  pro- 
tected them  against  smallpox;  nor  was  he  the  first  deliberately  to  in- 
oculate persons  with  cowpox  virus,  as  this  method  had  been  practised 
sporadically  before  his  time.  Jenner  was,  however,  the  first  medical 
man  to  give  the  matter  serious  thought  and  consideration,  and  to  test  the 
method  as  thoroughly  and  scientifically  as  it  was  possible  to  do  at  that 
period.  Thus  he  inoculated  with  smallpox  virus  those  whom  he  had 
previously  vaccinated  with  cowpox  virus,  and  found  them  immune  to 
smallpox.  These  experiments  were  courageously  repeated,  until  a  great 
truth  was  established,  which  has  resulted  in  almost  completely  eradi- 
cating the  disease  from  those  countries  or  communities  where  vaccina-. 
tion  is  thoroughly  carried  out.  As  was  to  be  expected,  Jenner  met  with 
considerable  opposition,  and  this  is  readily  understood  when  it  is  re- 
membered that  even  today — one  hundred  and  eighteen  years  later — 
there  are  those  who  refuse  to  accept,  or  are  unable  to  realize,  the  great 
benefits  of  this  pioneer  work.  Jenner  could  not  explain  his  results;  he 
maintained  that  he  was  dealing  with  a  modified  form  of  smallpox.  We 
of  today  have  no  better  means  of  establishing  the  truth  of  the  efficiency 
of  cowpox  vaccination,  nor  have  we  improved  any  on  his  method.  The 
specific  germ  of  smallpox  is  still  undiscovered,  and  we  must  agree  with 
Jenner  that  cowpox  is  probably  a  modified  form  of  smallpox  and  prac- 
tically harmless,  the  virus  of  cowpox  being  the  virus  of  smallpox 
modified,  attenuated,  and  rendered  practically  innocuous  by  passage 
through  a  lower  animal. 

Nothing  further  of  importance  was  accomplished  during  the  follow- 
ing eighty  years,  until  the  next  and  even  greater  epoch  ushered  in  the 
discoveries  in  bacteriology  and  the  first  immunization  by  Pasteur  based 
on  scientific  reasoning.  The  chickens  around  Paris  were  being  destroyed 
by  a  virulent  intestinal  infection,  and  Pasteur  first  isolated  the  causative 


144  IMMUNITY. — THEORIES    OF   IMMUNITY 

microorganism,  a  minute  bacillus,  which  he  found  was  capable  of  pro- 
ducing the  disease  experimentally  in  healthy  chickens.  Quite  by  acci- 
dent, so  it  seemed,  he  discovered  that  cultures  of  this  bacillus  could,  by 
prolonged  cultivation  be  attenuated,  for  when  these  cultures  were  inocu- 
lated into  chickens,  the  fowls  did  not  die  or  suffer  any  ill  consequences; 
further,  and  what  was  of  the  utmost  importance,  when  these  same  chick- 
ens were  inoculated  with  virulent  cultures,  they  were  found  to  be  im- 
mune to  chicken  cholera.  Here,  then,  was  the  key  to  active  immuniza- 
tion in  the  prevention  of  disease,  and  Pasteur  possessed  the  genius  to 
realize  the  full  significance  of  his  discovery. 

Armed  with  this  knowledge  Pasteur,  and  his  assistants,  Roux  and 
Chamberland,  next  studied  anthrax,  an  infectious  disease  of  cattle  that 
was  causing  a  great  annual  loss  to  the  farmers  of  France,  and  the  bacillus 
of  which  was  among  the  first  pathogenic  microorganisms  to  be  discovered. 
It  was  found  that  prolonged  cultivation  of  this  bacillus  was  insufficient 
to  attenuate  the  cultures,  as  the  spores  were  highly  resistant  and  re- 
tained their  pathogenicity  under  extreme  circumstances  and  over  pro- 
longed periods  of  time. 

In  1880  Touissant  published  a  method  of  attenuating  the  bacilli  by 
heating  the  blood  of  an  infected  animal  to  55°  C.  for  a  few  minutes; 
later,  Chauveau  secured  similar  results  by  heating  fresh  cultures  for  a 
few  minutes  at  80°  C.  Both  methods  were  uncertain,  and  neither  safe 
nor  practical.  After  prolonged  experimentation  Pasteur  found  that 
cultivating  the  bacilli  at  the  relatively  high  temperature  of  from  42°  to 
43°  C.  resulted  in  gradual  attentuation,  and  if  this  cultivation  was  con- 
tinued, the  cultures  were  robbed  entirely  of  their  disease-producing 
power.  Further,  subcultures  of  these  growths  when  kept  at  37°  C. 
did  not  regain  their  original  virulence,  but  maintained  for  generations 
the  grade  of  attenuation  reached  in  the  original  culture  the  result  of 
cultivation  for  a  certain  number  of  days  at  the  higher  temperature.  In 
this  manner  Pasteur  was  able  to  control  to  some  extent  the  degree  of 
attenuation,  and  by  inoculating  first  a  highly  attenuated  and  later  a 
less  markedly  attenuated  culture  he  could  immunize  animals  against 
anthrax.  This  discovery  was  soon  amply  verified.  The  original  method 
is  practically  the  one  employed  today,  and  is  proving  of  considerable 
economic  value. 

Pasteur's  next  great  experiment  was  undertaken  for  the  relief  of 
rabies,  a  condition  in  which,  for  the  first  time,  he  came  to  deal  with  a 
disease  that  not  infrequently  affects  man.  His  success  and  the  results 
of  his  discovery  of  an  effective  means  of  immunization  against  hydro- 


HISTORIC  145 

phobia  were  even  greater  than  in  previous  experiments.  Here  he  was 
dealing  with  a  disease  of  unknown  etiology,  the  causative  agent  of  which 
he  could  not  cultivate  artificially,  but  which  he  sought  to  attenuate  by 
a  new  process — that  of  drying. 

Pasteur  first  established  that  the  virus  of  rabies  is  contained  within 
the  tissues  of  the  brain  and  spinal  cords  of  infected  animals. 

He  then  invented  a  method  of  inoculating  animals  by  making  sub- 
dural  injections  of  an  emulsion  of  these  tissues.  By  repeated  passage 
of  a  virus  through  a  number  of  rabbits  a  virus  of  fixed  pathogenic  power 
(virus  fixe)  was  obtained.  By  inoculating  rabbits  with  this  virus  and 
removing  their  spinal  cords  immediately  after  death  and  drying  these 
over  a  desiccating  agent  at  room  temperature,  he  found  that  he  could 
modify  the  virulence  of  the  virus  at  will,  depending  on  the  length  of  the 
period  of  drying.  By  emulsifying  small  portions  of  attenuated  spinal 
cord  in  salt  solutions  and  injecting  these  he  was  able  gradually  to  im- 
munize animals  against  rabies,  and  finally  he  applied  the  treatment 
successfully  to  the  prevention  of  rabies  in  the  human  being. 

Antirabic  vaccination  is  largely  responsible  for  extending  our  knowl- 
edge of  the  possibility  of  securing  immunization.  Pasteur  has  taught 
us  at  least  three  different  methods  for  modifying  a  virus  in  the  prep- 
aration of  a  vaccine,  and  that  each  disease,  being  itself  a  special 
entity,  having  its  own  characteristics,  must  be  dealt  with  along  special 
lines. 

These  discoveries  were  largely  empirical,  and  the  explanations  of 
their  mechanism  are  now  only  of  historic  interest.  It  was  not  until  1883, 
when  Metchnikoff  shed  light  upon  the  problems  of  immunity  by 
making  a  series  of  remarkable  studies  on  the  role  played  by  certain  of 
the  body-cells  in  overcoming  infection,  and  the  part  they  played  in  the 
processes  of  immunity  in  general,  that  the  world  was  given  a  glimpse 
into  the  dark  problems  of  immunity.  These  observations  were  soon 
followed  by  investigations  showing  the  importance  of  the  body  fluids,  and 
since  that  time  a  great  deal  of  work  has  been  done  upon  these  subjects. 
As  a  consequence,  a  large  amount  of  data  of  a  wholly  new  order  has 
accumulated,  accompanied  by  the  introduction  of  a  host  of  new  terms 
expressing  diverse  views  and  theories  advanced  by  individual  workers. 
Of  the  many  theories  advanced  from  time  to  time  to  explain  the  phenom- 
enon of  immunity,  two  have  claimed  the  most  attention:  one  ascribes 
protection  and  cure  to  the  activity  of  certain  body-cells;  this  is  known 
as  the  cellular  theory;  and  the  other  attributes  these  qualities  to  the  body- 
fluids — the  humoral  theory.  The  chief  exponent  of  the  former  is  Metch- 
10 


146  IMMUNITY. — THEORIES    OF    IMMUNITY 

nikoff,  with  his  theory  of  phagocytosis,  whereas  Ehrlich  is  the  father  and 
leader  of  the  latter,  with  that  marvelous  invention  of  human  ingenuity, 
the  side-chain  theory. 

THEORIES  OF  IMMUNITY 

The  earlier  hypotheses  advanced  by  various  investigators  are  now 
only  of  historic  interest,  as  in  the  light  of  subsequent  discoveries  and 
observations  they  have  failed  to  offer  adequate  explanations. 

Pasteur's  own  theory  and  explanation  of  the  mechanism  of  acquired 
immunity  sought  to  show  that  the  microorganisms  living  in  the  infected 
animal  used  up  scvme  substance  essential  to  their  existence,  so  that,  for 
lack  of  proper  nourishment,  the  microorganisms  were  eventually  forced 
to  retire,  the  soil  being  unfit  for  further  occupation.  This  was  known 
as  the  "exhaustion  theory" 

Chauveau  considered  it  more  probable  that  the  microorganisms, 
after  having  lived  in  the  body  of  an  infected  animal,  produced  sub- 
stances that,  accumulating  in  the  blood,  had  an  inhibitory  action  on  the 
bacteria  and  were  inimical  to  their  further  existence.  This  was  known 
as  the  "retention  theory,"  and  in  some  particulars  was  just  the  opposite 
of  the  exhaustion  theory. 

THE  THEORY  OF  PHAGOCYTOSIS 

In  1883,  when  Metchnikoff  showed  that  certain  of  the  body-cells, 
and,  particularly,  the  polynuclear  leukocytes,  were  active  in  the  defense 
of  the  human  body  against  invasion  by  microparasites,  real  light  was 
thrown  upon  the  unknown  problems  of  immunity.  Although  he  has 
since  amplified  his  theory,  as  new  theories  were  adduced  to  describe  the 
part  played  by  the  body-fluids  and  the  organisms  themselves,  yet  his 
theory  of  phagocytosis  remains  a  demonstrable  fact,  and  establishes  the 
important  role  of  cells  in  the  processes  of  immunity. 

According  to  this  theory,  certain  of  the  body-cells  are  able  to  ingest 
an  infecting  parasite,  a  red  corpuscle,  or  other  cell  in  the  same  manner 
as  an  ameba  ingests  a  food-particle,  and  to  dispose  of  it  by  intracellular 
digestion  through  the  agency  of  ferments  known  as  "cytases. "  To 
such  cells  Metchnikoff  applied  the  name  phagocyte,  as  he  likened  them 
to  scavengers,  i.  e.,  they  were  concerned  in  picking  up  and  disposing  of 
offensive  material,  both  living  and  dead. 

Various  body-cells  are  capable  of  becoming  phagocytes.  The  poly- 
nuclear  leukocytes  are  particularly  active  in  acute  infections,  and  have 
been  called  microphages.  Endothelial  cells,  mononuclear  leukocytes, 


THEORIES    OF    IMMUNITY  147 

and  embryonic  connective-tissue  cells  are  more  active  in  chronic  infec- 
tions and  have  been  designated  macrophages. 

Although  the  original  explanation  of  phagocytosis  was  quite  plain 
and  far  reaching,  subsequent  discoveries  by  the  adherents  of  the  "  hu- 
moral theory"  showed  the  potent  influence  of  the  body-fluids  upon  the 
process.  It  was  demonstrated  that  without  the  aid  of  these  fluids 
phagocytosis  is  almost  negligible.  Metchnikoff  early  recognized  this 
fact,  and  sought  to  explain  the  influence  of  the  body-fluids  by  assuming 
that  they  contained  "  stimulins, "  or  substances  that  stimulated  phagocy- 
tosis. It  was  likewise  shown  that  the  body-fluids  contained  antibodies 
and  were  antibacterial,  independent  of  cells.  Metchnikoff  recognizes 
the  existence  of  these  conditions,  but  claims  that  they  are  duetto  the 
soluble  products  of  phagocytic  cells,  and  thus  in  a  broader  sense  would 
maintain  the  importance  of  his  phagocytes. 

It  was  soon  observed  that  in  certain  infections  leukocytes  or  other 
cells,  instead  of  being  attracted  toward  the  seat  of  infection  by  some 
unknown  chemical  stimulus,  (positive  chemotaxis),  were  repelled,  or  at 
least  the  attraction  was  counterbalanced  or  did  not  exist  (negative 
chemotaxis).  While  a  satisfactory  explanation  of  these  phenomena  is 
still  lacking,  it  may  be  stated  that,  in  general,  the  degree  of  negative 
chemotaxis  is  in  proportion  to  the  virulence  of  the  microparasite. 

In  1903  Wright  and  Douglas,  and  Neufeld  and  Rimpau  threw  con- 
siderable light  upon  this  subject.  They  demonstrated  experimentally 
that  one  action  of  the  body-fluids  was  directed  against  the  microorgan- 
isms, lowering  their  resistance,  and,  making  them,  as  it  were,  more 
attractive  to  the  phagocytes,  the  process  of  phagocytosis  was.  facilitated. 
To  these  substances  the  name  of  "opsonins"  (from  opsono,  I  prepare  for) 
was  applied  by  Wright,  while  Neufeld  called  them  "bacteriotropins." 

The  leukocytes  are  not,  however,  entirely  passive  and  willing  to  wait 
until  their  prey  is  weakened  and  fully  prepared  for  their  attack.  In  the 
presence  of  an  infection  they  are  found  to  increase,  and  this  leukocytosis 
is  known  to  be  a  valuable  addition  to  the  defensive  forces.  They  prob- 
ably also  undergo  qualitative  changes,  which  increase  their  antibacterial 
power.  It  has  been  shown  that  opsonized  bacteria  attach  themselves 
to  the  protoplasm  of  the  leukocytes,  a  physiochemical  phenomenon 
that  occurs  regardless  of  whether  the  leukocyte  is  dead  or  alive,  although, 
of  course,  only  the  living  leukocyte  is  able  to  ingest  them. 

That  phagocytosis  is  a  potent  and  very  important  factor  in  the 
mechanism  of  recovery  from  certain  infections  is  generally  admitted, 
and  although  it  probably  has  not  the  far-reaching  significance  originally 


148  IMMUNITY. — THEORIES   OF   IMMUNITY 

attributed  to  it,  yet,  throughout  the  discussion  of  immunity  the  im- 
portance of  the  phagocyte  itself  is  emphasized.  This  theory  of  Metch- 
nikoff  is  treated  more  fully  in  the  chapter  on  Phagocytosis. 

SIDE-CHAIN  THEORY 

The  humoral  theory  of  immunity,  which  would  ascribe  the  power  to 
resist  infection  to  the  body-fluids,  may  be  said  to  have  had  its  origin  in 
1896,  when  Fodor  discovered  that  the  blood  of  the  rabbit  will  kill  an- 
thrax bacilli  in  the  test-tube,  independent  of  cells  and  phagocytosis. 
Later  Buchner  adopted  this  theory,  and  sought  to  explain  the  bacteri- 
cidal action  of  blood-serum  as  dependent  upon  a  special  constituent 
which* he  called  alexin. 

With  the  discovery,  in  1890,  of  antitoxins  by  von  Behring  and  Kit- 
asato,  the  theory  received  fresh  support,  and  while  an  effort  was  made 
to  demonstrate  that  antitoxins  were  of  paramount  importance  in  ac- 
quired immunity,  evidence  soon  accumulated  to  show  that  this  anti- 
toxic power  is  operative  only  in  a  few  diseases,  chiefly  in  diphtheria  and 
tetanus. 

Fresh  support  to  the  "humoral"  as  against  the  " cellular"  explana- 
tion of  immunity  was  given  by  Pfeiffer  in  1894,  with  the  discovery  that 
cholera  vibrios  introduced  into  the  peritoneal  cavity  of  a  guinea-pig 
previously  immunized  against  cholera  became  transformed  into  granules, 
and  ultimately  passed  into  complete  solution  (bacteriolysis),  apparently 
without  the  aid  of  cells.  Bordet  then  showed  that  this  phenomenon 
was  due  to  two  distinct  substances — one,  the  "sensitizing  substance," 
which  is  specific  and  exists  only  in  the  immune  serum,  acting  only  on  the 
bacteria  against  which  the  animal  was  immunized,  and  the  other  a  non- 
specific substance,  found  in  the  fresh  serum  of  practically  all  animals, 
and  to  which  he  gave  the  name  "alexin,"  and  which  was  later  renamed 
by  Ehrlich  and  called  "complement." 

Of  the  various  theories  offered  in  explanation  of  these  observations, 
the  suggestive,  fascinating,  though  highly  hypothetic  theory  of  Ehrlich, 
known  as  the  side-chain  theory,  has  been  most  widely  accepted  and 
adopted  to  explain  new  discoveries  as  they  were  made.  The  theory 
has,  indeed,  aided  investigators  in  making  new  discoveries.  Nevertheless 
the  contention  of  Bordet,  that  its  too  ready  acceptance  without  sufficient 
convincing  proof  has  retarded  investigation,  should  not  be  ignored. 

The  basis  of  this  theory,  as  originally  proposed,  bore  no  relation  to 
the  subject  of  immunity,  but  was  advanced  in  1885  to  explain  the  pro- 
cesses of  nutrition. 


THEORIES    OF   IMMUNITY  149 

Ehrlich  asserts  that  a  cell  has  two  important  functions:  The  first  is 
the  special  physiologic  function,  as  that  of  a  nerve-cell  to  conduct;  of  a 
gland-cell,  to  secrete,  etc.  The  second  function  is  that  of  nutrition, 
and  presides  over  the  processes  of  waste  and  repair.  Furthermore, 
each  of  the  molecules  composing  the  complex  cell  is  believed  to  possess 
these  two  functions,  i.  e.,  one  is  concerned  with  the  special  function  of 
the  molecule,  and  the  other,  the  more  important  functional  portion,  is 
concerned  in  the  nourishment  of  the  molecule. 

The  second  portion,  or  that  concerned  with  nutrition,  is  of  more 
importance  in  relation  to  the  problems  of  immunity.  Ehrlich  con- 
ceives this  as  consisting  of  a  special  executive  center  or  main  portion 
("Leistenkern"),  in  connection  with  which  there  are  nutritive  side- 
chains,  receptors,  or  haptines  ("Leitenketter"),  which  "seize,"  or  rather 
enter  into  chemical  combination  with,  suitable  food  atoms,  which  is 
followed  by  a  sort  of  digestive  or  absorptive  process,  whereby  the  food 
material  is  incorporated  in  the  molecule. 

The  function  of  "seizing"  molecules  of  food  from  the  surrounding 
tissues  implies  a  selective  action  or  chemical  affinity  between  food 
atoms  and  the  portion  of  a  cell  or  side-arm  for  which  it  has  a  chemical 
affinity,  for  we  cannot  conceive  that  all  atoms  that  circulate  in  the  blood 
and  lymph  are  suitable  for  all  cells  at  all  times. 

The  food  molecule  in  the  fluid  surrounding  the  cell  is  conceived  as 
possessing  a  special  or  haptophore  portion  for  union  with  the  side-arm 
of  a  cell  molecule,  and  when  brought  into  relation  with  one  of  the  side- 
arms  or  receptors  of  the  cell  molecule,  the  two  are  "anchored,"  or  unite, 
just  as  a  key  fits  a  lock.  The  second  stage  involves  a  process  that  may 
be  compared  to  digestion,  by  which  the  food  material  is  prepared  and 
absorbed,  in  whole  or  in  part,  into  the  molecule  of  protoplasm. 

These  processes,  therefore,  are  conceived  as  being  chemical  rather 
than  physical,  and  our  diagrammatic  representations  of  them  have  no 
necessary  or  actual  morphologic  basis.  One  is  quite  likely  to  regard  the 
main  central  portion  as  the  nucleus  of  a  cell,  and  the  side-arms  as  small 
morphologic  projections  resembling  the  prickles  of  certain  epidermal 
cells.  These  processes  are  concerned  with  each  molecule  of  a  cell,  the 
main  portion,  or  "  Leistungskern, "  being  conceived  as  diffusing  through 
the  nutritive  part  of  the  molecule,  and  the  side-arm  receptors,  or  "Lei- 
tenketter," as  numerous  atoms  or  groups  of  atoms,  each  of  which  has 
a  chemical  affinity  for  some  particular  food-substance  circulating  in  the 
body-fluids,  and  necessary  for  the  life  of  the  molecule  in  question. 

Later  this  theory  was  amplified  by  Ehrlich  to  explain  the  action  of 


150  IMMUNITY. — THEORIES   OF   IMMUNITY 

toxins  and  the  production  of  antitoxins.  It  assumes  that  the  side-arms 
to  a  cell  molecule  are  exceedingly  numerous,  not  only  because  nutritive 
substances  are  varied,  but  because  special  cells  also  possess  different 
and  special  side-chains,  which  anchor  pathologic  material.  When 
infection  occurs,  and  in  addition  to  toxins  the  physiologically  normal 
substances  are  brought  to  the  cells,  they  likewise  find  suitable  receptors 
in  practically  all  or  certain  cell  groups,  and  become  anchored,  causing 
more  or  less  damage  to  the  cells. 

Having  combined  with  the  side-arms  or  receptors  of  a  cell,  the  toxin 
may  be  sufficiently  potent  to  kill  the  cell,  and  if  a  large  number  of  cells  are 
so  injured,  symptoms  of  disease  present  themselves  and  death  of  the 
infected  host  may  follow.  On  the  other  hand,  although  the  cell  has  lost 
one  or  more  of  its  side-arms,  it  may  not  be  dead,  and  it  proceeds  at  once 
to  repair  the  damage  done.  According  to  Weigert's  "overproduction 
theory/'  nature  is  lavish  in  its  processes  of  repair,  and  the  cell  not  only 
replaces  the  lost  receptors,  but  produces  them  in  numbers;  the  excess 
receptors,  having  no  space  for  attachment  to  the  cell,  are  thrown  off 
into  the  blood-stream.  Each  of  these  cast-off  receptors  or  haptines 
possesses  the  same  structure  as  the  original  receptor.  These  free  re- 
ceptors, then,  are  capable  of  combining  chemically  with  their  antigen, 
neutralizing  the  antigen  and  rendering  it  innocuous.  In  diphtheria 
and  tetanus  the  antigen  is  largely  the  soluble  toxin  of  the  bacilli,  and  the 
antitoxins  are  these  cast-off  receptors  produced  as  a  result  of  the  stimu- 
lating action  of  'the  toxins  upon  the  cells.  This  excess  of  receptors  is 
made  by  repeatedly  injecting  a  horse  with  increasing  doses  of  diphtheria 
toxin.  By  injecting  this  receptor-laden  (antitoxin)  serum  into  one 
suffering  from  diphtheria,  the  receptors  unite  with  free  diphtheria  toxin 
and  thus  protect  the  body-cells. 

For  the  production  of  these  receptors,  or  antibodies,  as  they  are  now 
called,  it  is  necessary,  as  previously  stated,  that  the  antigen  enter  into 
chemical  combination  with  the  cell,  so  that  the  usual  illustrations 
showing  the  theoretic  union  of  antigen  and  side-arm  by  physical  contact 
alone  probably  do  not  correctly  portray  what  actually  occurs.  As 
Adami  points  out,  the  antigen  probably  enters  into  intimate  relation- 
ship with  the  cell,  and  the  continued  stimulation  of  its  presence  is  re- 
sponsible for  the  production  of  an  excess  of  receptors,  in  addition  to 
the  overproductive  tendencies  of  nature's  repair. 

It  is  also  necessary  that  the  antigen  possess  sufficient  toxic  power  at 
least  to  stimulate  the  cell,  for  otherwise  antibodies  may  not  be  produced. 
Food  material,  for  instance,  being  physiologic,  is  assimilated  by  the 


THEORIES    OF   IMMUNITY  151 

cells  without  stimulating  the  production  of  antibodies,  as  otherwise 
the  food  would  be  attacked  by  cast-off  receptors  and  rendered  useless 
before  it  reaches  cells,  the  process  ending  in  starvation  and  death. 

The  host  in  whom  certain  cells  with  special  receptors  for  a  given 
poison  are  present  will  make  use  of  these,  no  matter  how  the  pathologic 
agent  is  introduced.  This  affinity  is  well  illustrated  in  tetanus,  where 
the  effects  produced  are  dependent  to  a  large  extent  upon  the  selective 
affinity  of  the  toxin  for  nerve-cells. 

THE  THREE  ORDERS  OF  RECEPTORS  AND  CORRESPONDING  ANTIBODIES 

First  Order:  Antitoxin  and  Simple  Antiferments. — The  simplest 
receptor  of  the  cell  molecule  is  composed  of  a  single  arm  or  haptophore, 
for  union  with  the  haptophore  portion  of  a  food  molecule.  As  previously 
stated,  a  molecule  of  toxin  is  conceived  as  being  composed  of  two  por- 
tions— one,  the  haptophore,  for  union  with  the  side-arm  or  receptor  of  a 
cell  molecule,  and  the  second,  the  toxophore,  in  which  its  toxic  action 
resides. 

The  first  stage  of  intoxication  of  a  cell  produced  by  a  true  toxin 
consists  in  the  union  of  the  haptophore  portion  of  the  toxin  molecule 
to  a  receptor  or  side-arm  of  the  cell  molecule,  this  receptor  being  one 
that  fits  the  toxin  molecule  "like  a  key  fits  a  lock."  Each  molecule  of 
the  body-cell  has  innumerable  receptors,  of  which  only  a  certain  number 
are  suitable  for  a  particular  toxin.  The  toxin  molecule  is  now  anchored 
to  the  living  cell,  and,  as  animal  experiments  with  a  great  number  of 
toxins  show,  this  union  is  a  firm  and  enduring  one  (Fig.  39). 

So  long  as  the  union  lasts  the  side-chain  involved  cannot  exercise  its 
normal  nutritive  physiologic  function — the  taking  up  of  food-stuffs. 
Furthermore,  the  toxophore  group  of  the  toxin  molecule  may  now  exert 
an  injurious,  enzyme-like  action  on  the  protoplasm  of  the  cell,  with  the 
result  that  the  protoplasm  is  poisoned.  If  only  a  few  of  the  cell  receptors 
are  united  with  toxin  molecules,  or  if  the  toxin  is  of  low  toxicity,  the 
effects  on  the  cell  may  be  slight.  If  more  are  joined  to  the  molecule 
or  the  toxin  is  highly  poisonous,  the  whole  molecule,  and  finally  the  cell 
itself,  may  be  greatly  disturbed,  and  produce  marked  symptoms,  or  the 
host  may  be  destroyed. 

Since  the  receptors  joined  to  the  toxin  molecules  are  incapacitated 
or  destroyed,  the  damage  is  repaired  by  the  regeneration  of  new  recep- 
tors. According  to  the  reparative  principles  worked  out  by  Weigert, 
the  repair  is  not  a  simple  replacement  of  the  defect — the  compensation 
proceeds  far  beyond  the  necessary  limit;  indeed,  overcompensation  is 


IMMUNITY. — THEORIES    OF   IMMUNITY 


the  rule,  and  this  forms  the  basis  of  Ehrlich's  theory.  If,  after  repair 
has  taken  place,  new  quantities  of  toxin  are  administered  at  proper 
intervals  and  in  suitable  quantities,  the  side-chains  that  have  been  pro- 
duced by  the  regenerative  process  are  taken  up  anew  in  combination 
with  the  toxin,  and  so  again  the  process  of  regeneration  gives  rise  to  the 
formation  of  fresh  side-chains.  "The  lasting  and  ever-increasing 
regeneration  must  finally  reach  a  stage  at  which  such  an  excess  of  side- 


0 


G 


FIG.  39. — THEORETIC  FORMATION  OF  ANTITOXINS. 

The  central  white  area  represents  a  molecule  of  a  cell;  the  shaded  portion  repre- 
sents the  cell  itself;  the  surrounding  area  represents  the  body-fluids  about  the  cell. 

r,  A  receptor  of  the  molecule  (first  order] ;  A,  overproduction  of  receptors,  which 
are  being  cast  off;  A2,  a  cast-off  receptor  free  in  the  body-fluids — now  an  antitoxin; 
A3,  a  molecule  of  antitoxin  combination  with  a  toxin  molecule  T3.  A%  a  cast-off 
receptor  still  within  the  parent  cell;  T,  a  toxin  molecule  in  combination  with  the  re- 
ceptor of  a  cell  molecule;  T2,  a  toxin  molecule  free  in  the  body-fluids;  T3,  a  toxin 
molecule  in  combination  with  antitoxin;  T4,  a  molecule  of  toxoid  (toxophore  group 
lost). 


chains  is  produced  that,  to  use  a  trivial  expression,  the  side-chains  are 
present  in  too  great  a  quantity  for  the  cell  to  carry,  and  are,  after  the 
manner  of  a  secretion,  handed  over  as  a  needless  ballast  to  the  blood. 
Regarded  in  accordance  with  this  conception,  the  antitoxins  represent 
nothing  more  than  side-chains  reproduced  in  excess  during  regeneration, 
and  therefore  pushed  off  from  the  protoplasm  and  so  coming  to  exist  in  the 
free  state"  (Ehrlich). 


THEORIES    OF   IMMUNITY  153 

This  theory  explains  the  specificity  of  the  antitoxins  for  a  given 
toxin;  thus  the  latter  causes  specific  chemical  stimulation  of  the  cell, 
which  induces  the  formation  of  specific  side-chains,  —  the  cast-off  re- 
ceptors, —  which  are  capable  of  uniting  with  the  toxin  molecules  free  in 
the  body-fluids  and  thus  neutralizing  them;  they  are,  therefore,  called 
antitoxins. 

This  theory  also  explains  why  a  minute  quantity  of  toxin  is  capable 
of  stimulating  the  production  of  a  large  amount  of  antitoxin,  and  why 
the  production  of  antitoxin  persists  for  some  time.  The  toxin  molecule 
must  be  conceived  as  entering  into  the  protoplasm  of  a  body  molecule 
and  residing  there  for  some  time,  acting  as  a  stimulus  to  the  cell,  with 
consequent  production  of  antitoxin.  Diagrammatic 
representations  of  this  process  would  seem  to  show 
that  a  physical  union  exists  between  toxin  and  cell 
receptors,  resulting  in  the  destruction  of  receptor, 
which  drops  off  and  is  replaced  by  a  number  of  recep- 
tors that,  for  lack  of  space  for  attachment  to  the 

cell,    are    thrown   off    into    the    blood-stream.     In     FlG-      40.—  THEO- 

RETIC       STRUO 
reahty,  by  the  act  of  immunization  certain  cells  of         TUREOFAMOLE- 

the  body  become  converted  into  cells  that  .secrete  AN^ToxoiD°XIN 
specific  antitoxin,  and,  as  shown  by  Salmonson  and  1,  Toxin:  H, 
Madsen,  the  administration  of  pilocarpin,  which 


augments   the  secretion  of   most   glands,  also  pro-     receptors  of  cells  or 
.     .  .  ,   .  antitoxin:  T,  toxo- 

duces  in  immunized  animals  a  rapid  increase  in  the     phore  group. 

antitoxin  content  of  the  serum.     The  formation  of       .   >  Toxoid:  Same 

structure   as   toxin 

antitoxin  is  constantly  going  on,  and  so  throughout      molecule    except 
i  .     i    .1  ,-,      .  £    ,1  that  the  toxophore 

a  long  period  the  antitoxin  content  of  the  serum     group  is  lost. 

remains  nearly  constant. 

In  the  production  of  antitoxin  the  haptophore  group  is  the  essential 
and  important  portion  of  the  toxin  molecule.  Even  though  the  toxo- 
phore group  is  lost,  —  and  when  this  occurs  the  toxin  is  called  toxoid 
(Fig.  40),  —  the  haptophore  group  is  capable  of  uniting  with  receptors 
and  stimulates  the  production  of  antitoxin.  In  fact,  in  effecting  immu- 
nization with  powerful  toxins  it  may  be  necessary,  in  the  first  few  in- 
jections that  are  given,  to  convert  artificially  all  or  a  portion  of  the 
toxin  into  toxoid,  so  that  antitoxins  will  be  produced  that  will  protect 
the  animal  against  subsequent  overdoses  of  toxin. 

The  production  of  antitoxins  must,  in  keeping  with  this  theory,  be 
regarded  as  a  function  of  the  haptophore  group  of  the  toxin.  It  is  easy, 
therefore,  to  understand  why,  out  of  the  great  number  of  alkaloids, 


154  IMMUNITY. — THEORIES    OF    IMMUNITY 

none  is  in  a  position  to  cause  the  production  of  antitoxins,  for  alkaloids 
possess  no  haptophore  group  that  anchors  them  to  the  cells  of  organs. 
As  has  been  stated,  in  the  formation  of  antitoxin  the  haptophore 
group  of  the  toxin  molecule  is  the  essential  portion;  the  toxophore 
group  is  much  less  important,  and  during  immunization  the  symptoms 
of  illness  due  to  the  action  of  the  latter  group  are  not  essential  to  and 
play  no  part  in  the  production  of  antitoxin.  It  must  be  said,  however, 
that  a  toxin  molecule  with  an  intact  toxophore  group  is  more  stimulating 
than  a  toxoid  in  which  this  group  is  absent;  therefore,  in  artificially 
immunizing  horses  for  the  production  of  antitoxin,  after  the  first  few 
injections  increasing  amounts  of  toxin  are  administered. 

Antibodies  of  the  Second  Order  (Agglutinins  and  Precipitins).— 
As  new  discoveries  were  made,  Ehrlich  amplified  his  theory  of  the  for- 
mation of  antibodies,  but  always  upon  the  original  and  basic  conceptions 
as  just  set  forth. 

We  have  seen  that  the  simplest  molecules  of  food  substances,  toxins, 
and  ferments,  substances  really  in  solution,  are  anchored  to  molecules 
of  cell  protoplasm  by  means  of  the  simple  side-arms  of  the  latter.  When 
this  chemical  union  has  taken  place,  the  food  or  toxin  may  be  assimi- 
lated without  undergoing  any  further  change.  With  more  complex 
food  substances,  however,  some  preparatory  treatment  is  necessary  be- 
fore they  become  available  for  final  assimilation.  The  large  molecule 
may  readily  enough  be  anchored  to  the  molecule  of  the  cell,  but  it 
probably  requires  some  preparation  before  it  becomes  available  for  the 
nutrition  of  the  cell. 

Accordingly,  Ehrlich  assumed  that  the  body-cells  are  furnished  with 
another  order  of  side-chains  or  receptors  composed  of  two  portions; 
one  part  or  group  for  union  with  the  food  substance,  and  called  the  hapto- 
phore group;  and  the  second  portion,  called  the  toxophore  or  zymophore 
group,  in  which  the  special  function  of  the  receptor  resides. 

Similarly,  certain  pathogenic  agents  that  are  more  complex  than 
soluble  toxins  or  ferments  combine  with  receptors  of  this  kind.  One 
arm,  the  haptophore  group  of  the  receptor,  combines  with  the  hapto- 
phore portion  of  the  pathogenic  molecule,  and  then  the  second  or  toxo- 
phore portion  of  the  receptor  exerts  some  special  action  upon  the  at- 
tached molecule.  Receptors  or  haptines  of  this  nature  are  known  as 
receptors  of  the  second  order;  antibodies  of  the  same  structure,  pro- 
duced and  cast  off  into  the  blood-stream  as  the  result  of  toxic  injury 
and  stimulation  of  body-cells,  are  known  as  antibodies  of  the  second 
order. 


THEORIES    OF   IMMUNITY 


155 


Two  such  antibodies  are  well  known.  In  one  we  find  that  the  toxo- 
phore  group  of  the  antibody  causes  clumping  or  agglutination  of  its 
antigen,  or  the  agent  that  caused  its  production,  and  hence  this  antibody 
is  called  an  agglutinin.  In  typhoid  fever,  for  example,  the  bacillus  or 
one  of  its  more  complex  products  causes  the  production  of  an  antibody 
of  this  nature,  so  that  when  the  serum  of  a  typhoid  fever  patient  is 
mixed  with  the  bacilli,  the  latter  lose  their  motility  and  form  clumps  or 
agglutinated  masses.  This  phenomenon  was  first  observed  by  Gruber 
and  Durham,  and  was  applied  in  a  practical  way  to  the  diagnosis  of 


FIG.  41. — FORMATION  OF  AGGLUTININS  AND  PRECIPITINS. 

The  central  white  area  represents  a  molecule  of  a  cell;  the  shaded  portion  repre- 
sents the  cell  itself;  the  surrounding  area  represents  the  body-fluids  about  the  cell. 

R,  Receptor  of  the  molecule  (second  order)]  R2,  overproduction  of  receptors, 
which  are  being  cast  off;  A,  a  cast-off  receptor  which  now  constitutes  the  antibody; 
A,  A2,  agglutinins  in  combination  with  the  antigen  (bacilli). 

typhoid  fever  by  Widal  and  Griinbaum.  The  second  antibody  of  this 
class,  the  precipitins,  resemble  the  agglutinins  quite  closely  (Fig.  41). 
Kraus  discovered  that  if  a  bouillon  culture  of  the  typhoid  bacillus 
is  filtered  through  porcelain,  and  a  few  drops  of  serum  from  a  typhoid 
fever  patient  or  from  an  animal  immunized  by  injections  of  typhoid 
bacilli  are  added  to  a  small  quantity  of  the  bacilli-free  filtrate,  a  faint 
cloud  will  appear  resembling  in  some  respects  that  observed  at  the  line 
of  contact  between  nitric  acid  and  urine  that  contains  a  trace  of  albumin. 


156  IMMUNITY. — THEORIES    OF   IMMUNITY 

The  toxophore  portion  of  this  antibody,  therefore,  appears  to  coagulate 
or  precipitate  soluble  substances,  and,  accordingly,  the  antibody  is 
known  as  a  precipitin.  As  will  be  pointed  out  later,  various  protein 
substances,  such  as  blood-serum,  milk,  egg-albumin,  etc.,  may  cause  the 
production  of  specific  precipitins. 

Antibodies  of  the  Third  Order  (Hemolysins,  Bacteriolysins,  Cyto- 
toxins). — Still  more  complex  molecules  of  food  material  require  con- 
version into  simpler  substances  before  they  may  be  assimilated  by  the 
molecules  of  the  cell.  It  is  essential  that  they  undergo  a  sort  of  digestion, 
and  accordingly  Ehrlich  has  conceived  that  special  side-arms  or  re- 
ceptors exist  for  this  purpose,  these  being  composed  of  two  grasping 
portions,  or  haptophore  groups,  one  for  union  with  the  complex  food 
molecule,  the  second  for  union  with  a  special,  ferment-like  substance 
present  in  the  blood  and  called  complement.  The  receptor,  therefore, 
acts  simply  as  a  connecting  link  or  interbody  between  food  molecule 
and  complement,  bringing  the  two  into  relation  with  each  other  when 
the  food  molecule  is  rendered  soluble,  i.  e.,  undergoes  lysis. 

With  highly  organized  cell  material,  such  as  red  blood-corpuscles  or 
bacteria,  it  is  found  that  receptors  of  this  nature  bring  about  their  de- 
struction by  lysis  by  attaching  them  to  a  suitable  complement.  During 
infections  with  various  bacteria,  therefore,  we  find  that  numerous  anti- 
bodies are  produced.  If  the  bacteria  produce  soluble  toxins,  specific 
antitoxins  are  produced  to  counteract  the  effects  of  these;  other  prod- 
ucts stimulate  the  production  of  agglutinins  and  precipitins ;  still  other 
products  or  the  whole  cell  cause  the  production  of  antibodies,  which 
are  not  in  themselves  destructive;  but  which  have  the  specific  power  of 
combining  with  the  cell  and  bringing  about  its  lysis  or  destruction  by 
bringing  it  into  relation  with  the  ferment-like  complement.  It  is  only 
by  means  of  a  special  antibody  of  this  nature  that  a  complement  may  be 
united  with  the  pathogenic  agent,  i.  e.,  the  complement  itself  cannot 
act  directly  upon  the  cell,  but  must  be  united  by  means  of  the  antibody. 

Ehrlich  has  termed  an  antibody  of  this  nature  an  amboceptor,  or 
interbody.  In  structure,  amboceptors  are  believed  to  possess  two  com- 
bining or  grasping  portions:  one,  the  haptophore  or  antigenophore 
group,  for  union  with  the  cell;  the  other  the  complementophile  group, 
for  union  with  a  complement  (Fig.  42). 

The  lysins  (bacteriolysins,  hemolysins,  and  other  cytolysins)  are 
antibodies  of  this  order.  If,  for  example,  the  erythrocytes  of  one  animal 
are  injected  into  an  animal  of  a  different  species,  hemolysins  will  be  pro- 
duced, the  hemolysin  being  a  specific  hemolytic  amboceptor  that  will 


PHAGOCYTIC   AND   SIDE-CHAIN  THEORIES 


157 


unite  corpuscles  of  the  animal  used  in  the  injection  and  only  these  cells, 
with  a  complement,  and  thus  bring  about  their  solution  or  lysis.  If 
certain  bacteria  (e.  g.,  the  cholera  bacillus)  are  injected  into  an  animal, 
specific  bacteriolysins  (bacteriolytic  amboceptors)  will  be  produced. 
Similarly,  specific  amboceptors  are  produced  during  the  course  of  in- 
fections with  typhoid  bacilli,  and  are  largely  instrumental  in  combating 
and  overcoming  this  infection.  It  is  important  to  remember,  however, 
that  although  these  amboceptors  probably  prepare  their  antigens  for 


FIG.  42. — FORMATION  OF  CYTOLYSINS  (HEMOLYSINS,  BACTERIOLYSINS,  CYTOTOXINS). 

The  central  white  area  represents  a  molecule  of  a  cell;  the  shaded  portion  repre- 
sents the  cell  itself;  the  surrounding  area  represents  the  body-fluids  about  the  cell. 

R,  Receptor  of  the  molecule  (third  order) ;  R2,  overproduction  of  receptors,  which 
are  being  cast  off;  A,  a  cast-off  receptor  which  now  constitutes  the  antibody  or  am- 
boceptor;  C,  molecule  of  complement  free  in  the  body-cells  and  body-fluids;  A2 A4, 
amboceptors  in  combination  with  molecules  of  a  cell  (antigen)  and  a  complement; 
A3,  an  amboceptor  in  combination  with  a  molecule  of  a  cell.  The  cell  (antigen)  is 
now  said  to  be  sensitized.  Lysis  does  not  occur  because  a  complement  is  not  united. 

lysis,  or,  in  the  meaning  of  Bordet,  " sensitize"  them,  they  are  not  in 
themselves  lytic,  final  solution  of  the  antigen  being  accomplished  by  the 
ferment-like  substance — the  complement. 


COMPATIBILITY  OF  THE  PHAGOCYTIC  AND  SIDE-CHAIN  THEORIES 

When  we  seek  to  compare  the  theory  of  Metchnikoff  with  that  of 
Ehrlich,  we  find  that  they  differ  only  in  minor  details,  the  fundamental 


158  IMMUNITY. — THEORIES   OF   IMMUNITY 

principles  not  being  contradictory;  they  may,  rather,  be  regarded  as 
one  set  of  phenomena  viewed  from  different  aspects. 

Since  its  original  announcement,  Metchnikoff  has,  on  different  oc- 
casions, enlarged  upon  his  theory  to  meet  certain  discoveries,  made 
chiefly  by  adherents  of  Ehrlich's  theory,  showing  the  presence  of  sub- 
stances in  the  blood-serum  and  other  body-fluids  that  are  potent  in  the 
processes  of  immunity  independent  of  cells.  Metchnikoff  claims, 
however,  that  these  antibodies  are  derived  from  the  group  of  cells 
classified  as  phagocytes,  and  thus  would  preserve  the  primary  impor- 
tance of  his  theory.  Ehrlich,  on  the  other  hand,  while  not  denying  that 
these  cells  may  be  a  source  of  their  formation,  points  out  that  they  are 
not  necessarily  the  sole  or  supreme  source,  but  may  be  formed  by  the 
general  body-cells  or  by  special  groups  of  cells  possessing  a  selective 
affinity  for  the'pathogenic  agent. 

The  theory  of  Ehrlich  is  essentially  a  chemical  one,  and  maintains 
that  the  union  of  food  or  pathologic  material  with  cells  is  a  chemical 
union;  his  views,  therefore,  possess  that  degree  of  definiteness  necessary 
to  constitute  a  plausible  chemical  theory.  The  theory  of  Metchnikoff 
would  explain  processes  of  nutrition  and  immunity  as  largely  founded 
on  a  physical  basis,  and  is  therefore,  necessarily  more  general,  being 
largely  biologic  and  vitalistic. 

The  two  theories  differ  in  two  more  or  less  hypothetic  points:  (1) 
In  the  manner  by  which  material  enters  into  relation  with  cells,  and 
(2)  the  relative  importance  of  certain  cells  in  the  formation  of  anti- 
bodies. Otherwise  both  are  intimately  related,  in  that  phagocytosis  is 
unimportant  if  removed  from  the  influence  of  antibodies  in  the  body- 
fluids,  and  these  same  antibodies,  although  probably  formed  accord- 
ing to  Ehrlich's  theory,  are  derived  in  part  from  Metchnikoff's  phago- 
cytes. 

Phagocytosis,  whether  by  leukocytes,  endothelial  cells,  or  by  newly 
developed  connective-tissue  cells,  is  very  common,  and  is  obviously  a 
most  important  factor  in  the  destruction  of  pathogenic  bacteria  and  in 
the  cure  of  infectious  disease.  In  virulent  infections,  however,  phagocy- 
tosis may  not  be  apparent;  the  leukocytes  are  not  attracted,  and  those 
in  the  vicinity  undergo  dissolution.  Later  in  these  infections,  however, 
phagocytosis  may  become  apparent,  due,  according  to  Metchnikoff,  to 
the  " adaptation"  of  the  cells  to  the  products  of  the  invading  micro- 
organism, whereby  the  weak  or  negative  chemotaxis  is  converted  into  an 
active  positive  chemotaxis  with  vigorous  digestion.  This,  however,  is 
not  primarily  due  to  increased  digestive  capacity  of  the  phagocytes, 


PHAGOCYTIC    AND    SIDE-CHAIN    THEORIES  159 

but  to  an  increase  of  opsonins  in  the  body-fluids;  these  opsonins  prepare 
the  bacteria  for  digestion. 

The  original  phagocytic  theory  did  not  explain  the  destruction  of 
bacteria  within  the  living  tissues  without  the  intervention  of  leukocytes, 
and,  what  is  even  more  striking,  a  similar  destruction  occurring  in  vitro 
by  serum  and  other  body-fluids  totally  devoid  of  cells.  Bacteriolysis 
has  been  shown  to  be  due  to  two  different  substances — one,  a  thermo- 
labile,  ferment-like  body  called  "cytase"  by  Metchnikoff  and  "  comple- 
ment" by  Ehrlich,  and  the  other  a  more  specific  thermostabile  body 
called  "fixateur"  by  Metchnikoff  and  "amboceptor"  by  Ehrlich. 
These  substances  appear  to  play  an  important  role  in  certain  infections, 
as,  for  example,  in  typhoid  fever  and  cholera,  and  were  studied  mainly 
by  the  adherents  of  the  side-chain  theory.  Metchnikoff  recognized 
their  existence  and  significance,  but  endeavored  to  preserve  the  primary 
importance  of  the  phagocytic  theory  by  claiming  that  they  are  products 
of  the  group  of  cells  classified  as  phagocytes.  Ehrlich,  however,  while 
not  denying  that  these  cells  may  be  one  source,  holds  that  they  are  not 
necessarily  the  sole  source,  but  that  they  are  products  of  general  cellular 
activity  or  of  special  groups  of  cells  that  have  shown  a  combining  affinity 
for  the  antigens. 

For  example,  Metchnikoff  holds  that  there  are  but  two  comple- 
ments,— macrocytase  and  microcytase, — and  that  these  are  formed  by 
destruction  or  solution  of  macrophages  and  microphages.  Ehrlich  has 
shown  quite  conclusively  that  there  are  many  complements,  and  that 
these  are  the  excretory  products  of  leukocytes,  and  probably  of  other 
cells  as  well.  Ehrlich  teaches  also  that  specific  amboceptors  or  fixateurs 
may  be  products  of  various  body-cells  other  than  those  classified  as 
phagocytes,  and  Metchnikoff  recognizes  their  existence,  but  holds  that 
they  are  formed  and  discharged  solely  by  the  leukocytes  or  other  pha- 
gocytic cells.  Ehrlich  has  shown  the  manner  in  which  complement  and 
amboceptor  produce  bacteriolysis,  and  Metchnikoff  has  amplified  his 
theory  to  meet  these  observations,  to  the  extent  that  destruction  of 
bacteria  is  recognized  as  being  brought  about  either  intracellularly,  by 
the  digestive  action  of  the  leukocytes,  or  extracellularly,  by  the  enzyme- 
like  action  of  the  cytase,  or  complement,  working  through  the  inter- 
mediation of  the  fixateur  or  amboceptor,  and  that  cells  that  are  poten- 
tially phagocytic  give  origin  to  these  antibodies. 

Regarding  the  structure  of  toxins  and  the  action  of  antitoxins  the 
two  theories  are  divergent,  and  whereas  Metchnikoff  is  inconclusive, 
Ehrlich  presents  definite  conceptions  that  are  well  supported  by  experi- 


160  IMMUNITY. — THEORIES   OF   IMMUNITY 

mental  data.  Metchnikoff  maintains  that  it  is  the  cells  that  absorb 
the  "toxin"  that  furnish  the  antitoxin.  In  other  words,  the  enzymes, 
as  microcytase  and  macrocytase,  exert  their  action  not  only  upon  the 
more  complex  molecules  of  microorganisms,  but  also  upon  their  simpler 
toxins,  fixing  or  otherwise  altering  them  until  they  can  finally  be  de- 
stroyed. This  explanation  would  lead  us  to  conclude  that  the  nerve- 
cells  which  bind  the  tetanotoxin  are  capable  of  furnishing  antitoxin, 
whereas  experimental  observations  are  absolutely  opposed  to  this 
narrower  view.  Metchnikoff  also  maintains  that  antitoxin  acts  by 
stimulating  the  leukocytes  to  absorb  and  destroy  toxin,  whereas  Ehrlich 
has  clearly  shown  that  antitoxin,  by  combining  chemically  with  the 
toxin,  neutralizes  it,  a  process  that  may  be  shown  in  vitro  entirely  in- 
dependent of  cells. 

From  what  has  been  said  it  will  be  seen  that  the  two  theories  are  not 
essentially  divergent,  and  that  we  are  unwarranted  in  clinging  to  one 
view  to  the  absolute  exclusion  of  the  other.  The  question  rests  largely 
on  which  of  the  body-cells  are  most  active  in  forming  antibodies,  and 
also  on  a  recognition  of  the  role  played  by  phagocytosis  in  certain  in- 
fections, such  as  staphylococcus,  streptococcus,  and  pneumococcus  in- 
fections. Ehrlich  has  attempted  an  explanation  of  the  method  by  which 
body-cells  form  antibodies,  and  the  manner  in  which  these  antibodies 
overcome  their  antigens;  he  has  placed  both  processes  upon  a  chemical 
basis,  involving  no  one  particular  group  or  class  of  cells.  Metchnikoff, 
on  the  other  hand,  has  shown  the  important  role  played  by  phagocy- 
tosis in  many  infections,  and  claims  that  the  antibodies  in  the  cir- 
culating fluids  are  the  products  of  these  phagocytes;  he  places  immunity 
more  largely  upon  a  physical  basis. 

The  various  phenomena  of  immunity  cannot  be  ascribed  either  to 
the  activity  of  the  body-cells  or  to  the  body  fluids  alone,  to  the  total 
exclusion  of  the  other — both  are  intimately  concerned  in  the  various 
phases  of  immunity. 

It  is,  moreover,  becoming  more  obvious  that  too  little  attention  has 
been  paid  to  the  influence  of  the  microorganism  in  the  phenomena  of 
immunity  reactions.  It  is  important  to  recognize  that  some  bacteria 
are  apparently  able  to  immunize  themselves  against  the  combative 
forces  of  their  hosts,  as  is  demonstrated  by  the  manner  in  which  strepto- 
cocci and  pneumococci  protect  themselves  with  a  capsule  and  resist 
phagocytosis.  Virulent  strains  and  "resistant  races"  may  be  evolved 
in  this  manner.  This  has  been  demonstrated  by  Ehrlich  with  regard 
to  the  action  of  various  arsenical  compounds  on  protozoa,  work  that 


ANTIGENS  161 

finally  culminated  in  the  brilliant  discovery  of  salvarsan.  Thus  atoxyl 
may  not  kill  all  the  trypanosomes  in  an  infected  animal,  those  escaping 
acquiring  a  new  power  of  resistance  to  the  poison  and  become  atoxyl- 
resistant.  The  production  of  " resistant  races,"  not  only  among  the 
protozoa,  but  also  in  the  class  of  bacteria,  complicates  enormously  the 
practical  problems  of  immunity. 

ANTIGENS 

Briefly  defined,  antigens  are  substances  that  can  cause  the  formation 
and  appearance  of  antibodies  in  the  body -fluids. 

So  far  as  is  now  known,  antigens  are  colloids,  and  are  usually  protein 
in  nature.  Every  known  soluble  protein  may  in  some  degree  act  as  an 
antigen,  and  recent  investigations  would  seem  to  show,  although  they 
do  not  definitely  prove,  that  toxic  glucosids  and  various  lipoids  may  to 
some  extent  act  in  this  same  capacity  The  protein  antigens  may  be 
quite  varied:  thus  antibodies  are  produced  not  only  by  the  injection 
of  bacteria  or  their  toxins,  but  also  by  erythrocytes,  serums  of  different 
animals,  egg-albumen,  milk,  etc. 

Of  the  cleavage  products  of  proteins,  it  is  certain  that  none  of  the 
amino-acids  and  simple  polypeptids  can  act  as  antigens;  there  is,  how- 
ever, some  evidence  to  show  that  the  proteoses  possess  antigenic  proper- 
ties. It  has  been  shown  by  Gay  and  Robertson1  that  if  the  antigenic 
cleavage  products  of  casein  are  resynthesized  by  the  reverse  action  of 
pepsin  into  a  protein  resembling  paranuclein,  this  synthetic  protein  is 
capable  of  acting  as  an  antigen.  Protamins  and  globin  were  found  to 
be  non-antigenic,  although  globin  combined  with  casein  formed  a  com- 
pound of  antigenic  power  in  that  it  produced  an  antibody  yielding 
complement-fixation  reactions  with  globin. 

Whether  the  entire  protein  molecule,  or  only  groups  thereof,  deter- 
mine the  characteristics  of  the  antigen  and  the  antibody  is  not  definitely 
known.  Wells  and  Osborne2  have  recently  submitted  evidence  showing 
that  a  single  protein  molecule  can  act  as  an  antigen  and  produce  more 
than  one  antibody. 

Non-protein  Antigens. — Ford3    was  able  to  immunize  rabbits  by 
injecting  a  toxic  glucosid  contained  in  extracts  of  Amanita  phalloides, 
producing  an  antibody  antihemolytic  for  the  hemolysin  of  Amanita 
when  diluted  1 :1000.     Abderhalden  and  others  have,  found  that  specific  % 
enzymotic  substances  appear  in  the  blood  of  animals  injected  with  car- 

1  Jour.  Biol.  Chem.,  1912, 12,  233;  Jour.  Exp.  Med.,  1912, 16,  479;  1913, 17,  535. 

2  Jour.  Infect.  Dis.,  1913,  12,  341.  8  Jour.  Infect.  Dis.,  1907,  4,  541. 

11 


162  IMMUNITY. — THEORIES   OF   IMMUNITY 

bohydrates  and  fats.  Recent  developments  in  immunologic  research 
would  indicate,  therefore,  that  a  complex  toxic  glucosid  that  can  be 
hydrolyzed  by  enzymes  may  act  as  an  antigen. 

The  intimate  relationship  of  lipoids  to  complement  fixation  reactions, 
especially  in  syphilis,  has  naturally  led  to  investigations  regarding  the 
possibility  of  lipoids  acting  as  true  antigens.  In  testing  for  the  Wasser- 
mann  reaction  the  use  of  lipoids  in  the  form  of  tissue  extracts  to  serve 
as  an  antigen  does  not  mean  that  it  is  a  true  antigen;  in  fact,  experi- 
mental work  indicates  quite  strongly  that  these  lipoidal  substances  are 
incapable  of  producing  antibodies  when  injected  into  animals. 

Much  and  others  have  worked  with  lipoids  secured  from  a  strepto- 
thrix,  and  which  is  called  "nastin,"  and  they  assert  that  this  substance 
may  be  used  in  immunizing  animals  with  the  production  of  an  antibody 
yielding  complement  fixation,  with  nastin  as  the  antigen.  Similar 
results  have  been  described  for  the  fatty  substances  from  tubercle  bacilli 
("tuberculonastin").  Kleinschmidt l  accepts  the  antigenic  'nature  of 
nastin  in  reactions,  but  was  unable  to  secure  antibodies  by  immunizing 
rabbits  with  this  substance. 

Ritchie  and  Miller2  could  demonstrate  no  antigenic  activity  in  the 
lipoids  of  serum  or  corpuscles.  Thiele3  calls  attention  to  the  fact  that 
lipoids  possess  no  specificity,  and  that  they  cannot  act  as  antigens  with 
the  production  of  antibodies.  On  the  other  hand,  Meyer 4  has  reported 
the  production  of  specific  complement-fixation  antibodies  by  immuniz- 
ing rabbits  with  acetone-insoluble  lipoidal  substances  obtained  from 
various  teniae.  He  has  also  found  the  acetone-insoluble  fraction  of 
tubercle  bacilli  to  serve  as  antigens  in  complement-fixation  reactions 
with  antibodies  of  the  tubercle  bacillus,  and  much  more  effectively  than 
with  the  protein  residue  of  the  bacilli.  Beigel5  has  observed  that  after 
injecting  lecithin  in  rabbits  an  increase  occurs  in  the  lipase  content  of 
the  blood  and  tissues,  with  the  presence  of  complement-fixing  anti- 
bodies, and  Jobling  and  Bull6  have  found  an  increase  in  serum  lipase 
after  immunizing  with  red  corpuscles. 

It  will  be  noticed,  therefore,  that  the  results  of  various  investigations 
regarding  the  true  antigenic  properties  of  lipoids  are  not  in  accord. 
It  should  be  emphasized  that  the  complement-fixation  reaction  does  not 
constitute  a  reliable  index  to  the  study  of  this  problem,  as  so  little  is 

1  Berl.  klin.  Wochschr.,  1910,  47,  57.         2  Jour.  Path,  and  Bact.,  1913,  17,  427. 

3  Zeit.  f.  Immunitat.,  1913,  16,  160. 

4  Zeit.  f.  Immunitat.,  1910,  7,  732;  1911,  9,  530;  1912,  16,  355. 
6  Deut.  Arch,  f .  klin.  Med.,  1912,  106,  47. 

6  Jour.  Exper.  Med.,  1912,  16,  483. 


ANTIBODIES  163 

understood  of  the  actual  nature  of  this  reaction  itself.  That  lipoids 
serve  a  very  important  purpose  in  the  absorption  or  fixation  of  comple- 
ment in  vitro,  as  is  so  well  demonstrated  in  Wassermann's  reaction  for 
syphilis,  is  undoubtedly  true,  but  this  does  not  indicate  that  the  anti- 
body in  the  blood-serum  of  syphilitics  is  in  the  nature  of  a  true  lipoid 
antibody,  and,  indeed,  investigation  on  this  subject  would  seem  to 
indicate  that  it  is  not.1 

It  will  be  understood,  therefore,  that  the  question  of  substances  other 
than  proteins  acting  as  true  antigens  must  be  regarded  as  an  open  one, 
requiring  further  investigation.  The  relation  of  proteins,  however,  to 
the  production  of  antibodies  has  been  fully  established,  and  is  at  present 
receiving  renewed  attention  through  the  researches  of  Vaughan  and  his 
coworkers  and  Abderhalden.  As  has  been  stated  in  a  previous  chapter, 
Vaughan  regards  the  protein  constituents  of  bacterial  and  other  cells  as 
the  main  antigenic  principle  capable  of  causing  the  production  of  specific 
proteolytic  ferments,  which  split  the  new  bacterial  protein,  releasing  a 
toxic  product  responsible  for  the  symptoms  and  lesions  of  the  infection. 
Abderhalden  has  also  demonstrated  the  presence  of  proteolytic  ferments 
in  the  blood-serum  after  experimental  immunization  with  proteins,  and 
in  the  serum  of  pregnant  women,  due  to  the  antigenic  stimulation  of 
syncytial  cells,  capable  of  splitting  their  substrata  in  vitro  into  amino- 
acids  and  other  simple  cleavage-products.  These  investigations  serve 
to  show  the  intimate  relation  that  proteins  bear  to  the  problems  of 
infection  and  immunity,  and  demonstrate  that  antibodies  may  be  largely 
in  the  nature  of  ferments,  and  that  immunologic  reactions,  both  in  the 
living  tissues  and  in  the  test-tube,  are  largely  in  the  nature  of  disin- 
tegrative  enzymic  processes. 

ANTIBODIES 

The  term  antibody  is  used  to  designate  the  specific  bodies  produced  by 
the  cells  of  a  host  in  reaction  against  an  antigen,  as  an  infecting  micropara- 
site  and  its  products  or  other  foreign  protein. 

Various  kinds  of  antibodies  may  be  produced  by  the  same  antigen 
and  by  different  antigens.  Some  neutralize  the  soluble  toxin  of  the 
antigen  (antitoxin);  others  agglutinate  or  precipitate  their  antigens 
(agglutinins  and  precipitins) ; .  still  others  cause  complete  dissolution  of 
the  antigen  (hemolysins,  bacteriolysins,  etc.),  and  others  again  may  so 
alter  the  antigen  and  lower  its  resistance  as  to  render  it  more  easily 
phagocytable  by  the  body-cells  (opsonins  or  bacteriotropins) . 

1  Further  discussion  on  the  question  of  lipoids  acting  as  antigens  will  be  found 
in  Chapter  XXVII  in  a  consideration  of  anaphylactogens. 


164  IMMUNITY. — THEORIES    OF    IMMUNITY 

Tissues  Concerned  in  the  Production  of  Antibodies. — A  large  amount 
of  experimental  work  has  been  conducted  in  the  study  of  the  problem 
of  where  in  the  body  the  antibodies  are  formed  that  develop  in  response 
to  immunization.  The  recent  investigations  of  Hektoen  and  Curtis,1  who 
studied  the  effect  on  antibody  production  of  the  removal  of  various 
organs,  and  of  Hektoen2  and  Simonds  and  Jones,8  on  the  influence  of 
exposure  to  z-rays,  and  of  Simonds  and  Jones,4  on  the  effect  of  injections 
of  benzol,  indicate  that  the  mechanisms  concerned  for  the  production 
of  antibodies  are  quite  secure  from  certain  disturbances  and  are  princi- 
pally located  in  the  leukocytes  and  blood-forming  organs,  as  the  spleen, 
lymphatic  tissues,  and  bone-marrow. 

Specificity  of  Antibodies. — Antibodies  are  usually  specific  for  their 
antigen,  and  it  is  upon  this  general  law  that  the  reactions  of  immunity  are 
based.  It  should  be  remembered,  however,  that  not  all  antibodies  are 
protective;  the  agglutinins,  for  instance,  apparently  do  not  injure  their 
antigen.  On  the  other  hand,  an  animal  may  enjoy  an  immunity  with- 
out demonstrating  the  presence  of  any  antibody  in  the  body-fluids,  and 
another  animal  may  show  antibodies  generally  considered  as  possessing 
protective  powers,  as,  for  example,  the  bacteriolysins,  without  neces- 
sarily being  immune. 

Upon  what  does  the  specificity  of  antibodies  and  immunologic 
reactions  depend?  Specificity  was  at  first  believed  to  depend  solely 
upon  some  peculiar  biologic  relationship  of  the  antigens,  for  it  was  found 
comparatively  easy  to  differentiate  the  serum  of  animals  of  dissimilar 
nature  by  means  of  the  precipitin  and  other  reactions,  and,  as  serum  pro- 
teins, which  seemed  to  be  quite  similar  chemically,  but  which  were 
obtained  from  unrelated  species,  were  sharply  differentiated  by  the 
biologic  reactions,  it  was  considered  that  the  specificity  must  be  depend- 
ent upon  some  principle  quite  apart  from  the  ordinary  chemical  sub- 
stances. 

With  the  use  of  proteins  other  than  serums,  and  especially  when  more 
or  less  purified  proteins  were  employed,  it  has  been  quite  firmly  estab- 
lished that  specificity  depends  upon  chemical  composition,  and  that 
differences  in  species,  as  exhibited  by  their  biologic  reactions,  depend  upon 
distinct  differences  in  the  chemistry  of  their  proteins  (Wells). 

Pick  and  his  coworkers  have  shown  that  two  kinds  of  specificity 
exist  in  each  protein  molecule:  (1)  One  of  these  is  easily  changed  by 
various  physical  agents,  such  as  heat,  cold,  and  partial  coagulation. 

1  Jour.  Infect.  Dis.,  1915,  17,  409.  2  Jour.  Infect.  Dis.,  1915,  17,  415. 

3  Jour.  Med.  Research,  1915,  33,  183.         4  Jour.  Med.  Research,  1915,  33,  197. 


ANTIBODIES  165 

When  an  antigen  is  altered  by  heat,  it  produces  an  antibody  that  reacts 
best  with  the  heated  antigen;  heating  does  not,  however,  destroy  the 
characteristics  of  the  antigen  of  this  species,  as  its  antibody  will  not  react 
with  the  heated  antigen  of  another  species.  (2)  The  second  alteration 
involves  a  profound  chemical  change  ,of  the  antigen,  whereby  it  is  so 
altered  that  it  loses  the  characteristics  peculiar  to  the  species,  and  pro- 
duces an  antibody  that  will  react  with  the  altered  antigen,  but  not  with 
the  unaltered  antigen,  even  from  the  same  animal.  For  example,  it  is 
possible  so  to  alter  the  serum  protein  of  a  rabbit  by  treatment  with 
nitric  acid  that  the  nitroprotein  injected  back  into  the  same  rabbit  will 
produce  an  antibody  specific  for  the  nitroprotein,  but  which  does  not 
react  with  the  unchanged  serum  protein.  These  changes  are  apparently 
closely  related  to  the  aromatic  radicals  of  the  protein  antigen,  for  they 


HEMOLYSINS 
k     BACTERIOLY5IW5 
C\     BACTERIOTROPINS 
CYTOTOWN5 


FIG.  43. — GENERAL  SCHEME  OF  ANTIGENS  AND  ANTIBODIES. 

Antitoxins  and  antif erments :  R,  Receptor  of  a  molecule  of  a  cell;  T,  a  toxin 
molecule;  t,  toxophore  group  of  the  toxin  molecule;  h,  haptophore  group  of  the  toxin 
molecule;  A,  cast-off  receptor  and  constitutes  antitoxin. 

Agglutinins  and  precipitins:  A.  R,  Receptor  of  cell  with  antigen  attached;  B, 
a  bacterial  molecule  (antigen)  attached  to  a  receptor;  A.  or  P,  an  agglutinin  or  pne- 
cipitin;  h,  haptophore  group  of  the  antibody;  a,  agglutinophore  group  of  an  agglu- 
tinin. 

Hemolysins,  etc.:  A,  Cast-off  amboceptor  (hemolysins,  bacteriolysin,  etc.); 
h,  haptophore  group  of  amboceptor;  c,  complementophile  group;  C,  molecule  of 
complement. 

are  effected  by  introducing  into  the  protein  molecules  substances  that 
are  known  to  combine  with  the  benzine  ring,  e.  g.,  iodin,  diazo-  and  nitro- 
groups.  Pick,  appreciating  the  fact  that  the  number  of  different  aromatic 
radicals  in  the  protein  molecule  are  limited,  interprets  the  significance 
of  these  radicals  as  depending  upon  their  arrangement,  rather  than  upon 
their  number,  in  the  protein  molecule.  Granting  the  number  of  possible 


166 


IMMUNITY. — THEORIES    OF    IMMUNITY 


variations  in  the  arrangement  of  the  amino-acids  in  a  protein  molecule 
which  the  great  number  of  these  radicals  provides,  there  is  no  difficulty 
in  understanding  the  possibility  of  an  almost  limitless  number  of  specific 
distinctions  between  proteins. 

It  may  be  stated,  however,  in  general,  that  immunologic  reactions, 
such  as  that  of  anaphylaxis,  are  as  delicate  in  distinguishing  between 
proteins  as  are  chemical  analyses.  Distinctions  may  be  made  by  these 
reactions  with  quantities  too  small  for  making  accurate  chemical  de- 
terminations. 

In  succeeding  chapters  we  shall  consider,  first,  the  different  kinds  of 
immunity,  as  dependent  upon  the  presence  of  various  factors,  and  then 
the  role  played  by  the  phagocytes  and  the  body-fluids  in  immunity, 
with  a  more  detailed  consideration  of  the  various  antibodies. 

It  may  be  useful  here  to  draw  up  in  tabular  form  a  list  of  the  various 
antigens  and  antibodies  with  which  we  are  mainly  interested  in  that 
portion  of  immunity  involving  infection  with  vegetable  or  animal  para- 
sites, and  the  products  of  their  metabolism  or  degeneration  (Fig.  43). 


ANTIGENS 
Toxins: 

1.  Soluble    bacterial    toxins    (diph- 

theria and  tetanus  toxins,  etc.). 

2.  Phyto-  (vegetable)  toxins  (ricin, 

abrin,  etc.). 

3.  Simple     zoo-     (animal)     toxins; 

(snake,   spider,   toad  venoms). 

4.  Complex    zootoxins,     as    snake 

venom,  requiring  complement 
for  action. 

Enzymes  or  ferments  (rennin,  lipase, 
etc.). 

Precipitogenous  substances  (soluble  ani- 
mal and  vegetable  proteins). 

Agglutinogenous  substances  (bacteria, 
erythrocytes,  etc.). 

Opsonigenpus  substances  (bacterial  en- 
dotoxins  or  aggressins?). 

Cytolignequs  substances: 

1.  Vegetable  cells  (bacteria). 

2.  Animal  cells  (erythrocytes,  sper- 

matozoa,  kidney  tissue,   etc.). 


ANTIBODIES 
Antitoxins : 

1.  Antitoxins        (diphtheria       and 

tetanus  antitoxins,  etc.). 

2.  Anti-  (phyto-)  toxins  (antiricin, 

antiabrin,  etc.). 

3.  Anti-  (zoo)  toxins  (antivenins). 

4.  Antihemolysins,  etc. 


Anti-enzymes     (antirennin,     antilipase, 

etc.). 
Precipitins. 

Agglutinins. 

Opsonins  (acting  singly  or  with  comple- 
ment) . 
Cytolysins: 

1.  Bacteriolysins. 

2.  Hemolysins,    spermatolysins, 

nephrolysins,  etc. 


CHAPTER  IX 

THE  VARIOUS  TYPES  OF  IMMUNITY 

As  has  been  stated  in  the  preceding  chapter,  it  is  generally  agreed 
that  various  antibodies  and  other  protective  agencies  exist,  although 
opinions  differ  as  to  the  source  and  relative  importance  of  these  to  re- 
sistance to  and  recovery  from  various  infections.  Whether  or  not  a 
particular  antibody  is  derived  from  a  certain  group  of  cells  is  largely  a 
matter  of  individual  opinion,  because  of  the  difficulty  of  deciding  the 
point  by  actual  experimental  evidence.  Of  far  more  importance  is  a 
knowledge  of  the  properties  of  antibodies  and  of  the  role  they  may  play 
in  the  processes  of  immunity.  It  is  seldom  that  resistance  to,  or  re- 
covery from,  an  infection  is  dependent  upon  one  defensive  factor: 
usually  several  agencies  are  operative,  although  one  factor  may  pre- 
dominate. For  example,  antitoxins  are  known  to  neutralize  their 
respective  toxins,  and  are  of  most  value  in  combating  the  toxemias, 
such  as  diphtheria  and  tetanus;  bacteriolysins  cause  the  death  of  and 
may  totally  destroy  their  antigens,  and  play  an  important  part  in  the 
recovery  from  infections  with  bacilli  of  the  typhoid-colon  and  cholera 
groups;  phagocytosis  in  itself  is  of  importance  in  staphylococcus  in- 
fections, and  is  of  primary  importance,  in  conjunction  with  the  opsonins, 
in  recovery  from  pyogenic  infections  in  general;  agglutinins  and  pre- 
cipitins  do  not  appear  to  have  a  direct  inimical  influence  on  their 
antigens,  but  are  probably  secondary  factors,  and  contribute  in  some 
manner  toward  their  destruction.  Along  with  important  non-specific 
factors,  these  various  antibodies  are  responsible  for  the  different  forms 
of  immunity,  which  may  now  be  considered  in  their  more  general  as- 
pects. 

There  are  two  forms  of  immunity — natural  and  acquired. 

NATURAL  IMMUNITY 

Natural  immunity  is  the  resistance  to  infection  normally  possessed, 
usually  as  the  result  of  inheritance,  by  certain  individuals  or  species  under 
natural  conditions. 

The  mechanism  of  this  type  of  immunity  is  very  complex,  and  bears 
an  intimate  relation  to  the  subject  of  infection,  both  local  and  general, 

167 


168  THE   VARIOUS   TYPES   OF   IMMUNITY 

the  nature  of  the  infecting  parasite,  and  the  presence  or  absence  of 
specific  antibodies  in  the  body-fluids.  In  many  instances  this  type  of 
immunity  is  dependent  upon  non-specific  causes — is  frequently  relative 
and  seldom  absolute.  For  example,  fowls  are  immune  to  what  may  be 
called  an  ordinary  dose  of  tetanus  toxin,  but  succumb  readily  to  larger 
doses;  rats  are  highly  immune  to  diphtheria  toxin,  and  readily  withstand 
the  effects  of  an  amount  equaling  1000  lethal  doses  for  a  guinea-pig,  but 
still  larger  doses  may  prove  fatal;  hedgehogs  possess  complete  or  almost 
complete  immunity  for  the  amount  of  snake  venom  deposited  in  an 
ordinary  strike,  but  if  the  venoms  of  several  snakes  are  collected  and 
injected  at  one  time,  the  result  is  fatal. 

Species  immunity  is  a  type  of  natural  immunity,  best  illustrated  by 
the  immunity  of  man  to  certain  diseases  of  the  lower  animals,  such  as 
fowl  cholera,  swine-plague,  distemper,  Texas  cattle  fever,  mouse  septi- 
cemia,  etc.;  and,  conversely,  by  the  immunity  of  animals  to  diseases 
common  to  man,  such  as  measles,  cholera,  typhoid  fever,  scarlet  fever, 
chicken-pox,  etc.  Although  the  close  relation  of  man  to  the  domestic 
animals  furnishes  ample  opportunity  for  infection,  yet  a  complete 
immunity  is  frequently  observed. 

Racial  immunity  is  that  type  of  natural  immunity  existing  among 
members  of  the  same  species.  For  example,  negroes  are  believed  to 
enjoy  immunity  to  yellow  fever  and  Mongolians  to  scarlet  fever.  As 
a  matter  of  fact,  well-marked  examples  of  racial  immunity  are  extremely 
rare,  as  not  infrequently  the  disease  in  question  may  have  been  acquired 
in  early  infancy  in  a  clinically  unrecognized  form. 

Similarly,  close  biologic  relationship  is  no  guarantee  that  animals 
will  behave  alike  toward  infection.  For  example,  the  white  mouse  is 
immune  to  glanders,  the  house  mouse  is  somewhat  susceptible,  and  the 
field  mouse  is  highly  susceptible.  The  rabbit,  guinea-pig,  and  rat  are 
rodents,  but  though  the  rabbit  and  the  guinea-pig  are  susceptible  to 
anthrax,  the  rat  is  largely  immune.  Mosquitos,  though  closely  re- 
lated, behave  differently  toward,  the  malarial  parasite.  The  Culex 
does  not  carry  the  parasite  at  all,  and  of  the  Anopheles,  one  species, 
Anopheles  maculipennis,  is  quite  susceptible  and  well  recognized  as  a 
carrier  of  the  parasite,  whereas  Anopheles  punctipennis,  though  closely 
related,  is  not  susceptible  to  it. 

Examples  of  individual  immunity,  while  not  infrequent,  are  not  con- 
stant and  seldom  absolute.  Certain  persons  appear  to  possess  a  defi- 
nite immunity  to  scarlet  fever  and  diphtheria,  although  they  may  be 
freely  exposed;  others  may  pass  through  various  epidemics  of  other 


NATURAL   IMMUNITY  169 

infectious  diseases,  such  as  measles,  pertussis,  etc.,  without  becoming 
infected.  I  have  noticed,  on  several  occasions,  that  resident  physicians, 
on  service  in  scarlet-fever  wards  for  many  months  or  years,  having 
escaped  infection  though  brought  in  intimate  contact  with  severe  forms 
of  the  disease,  finally  contracted  the  disease  upon  returning  from  a  short 
vacation. 

Natural  immunity  may  be  due  to  the  following  causes: 

1.  Various  non-specific  factors  may  prevent  infection;    among  these 
may  be  mentioned — (a)  The  integrity  of  the  epithelium  of  the  skin  and 
mucous  membranes,  and  (6)  the  chemical  and  physical  action  of  various 
secretions,  such  as  the  gastric  fluid,  the  intestinal  juices,  and  the  saliva. 

2.  A  particular  route  for  the  introduction  of  infecting  microparasites 
may  be  necessary.     For  example,  intestinal  diseases,  such  as  typhoid 
fever  and  cholera,  are  usually  due  directly  to  swallowing  of  the  infecting 
microorganisms,  infection  in  this  type  of  disease  seldom,  if  ever,  occurring 
through  the  skin.     This  is  probably  due  in  part  to  the  lowered  vitality 
of  the  intestinal  mucosa,  together  with  a  peculiar  selective  affinity  of 
the  bacteria  for  the  cells  of  these  tissues,  aided  by — (a)  the  biologic 
nature  of  the  invading  bacterium,  which  grows  best  under  the  more 
favorable  cultural  conditions  of  the  intestinal  canal.     This  selective 
action  is  further  illustrated  by  the  tendency  of  dysentery  toxin  to  attack 
the  intestinal  mucosa  when  the  bacilli  or  toxin  is  administered  intra- 
venously. 

3.  Certain  tissues  appear  to  possess  a  marked  local  immunity  to  certain 
bacteria.     In  considering  examples  of  local  immunity,  various  factors, 
such  as  the  question  of  exposure,  the  thickness  of  the  epidermis,  and  the 
kind  and  quantity  of  the  local  secretions  must  be  borne  in  mind. 
For  example,  Trichina  spiralis  affects  the  muscles,  never  the  bones, 
and  but  rarely  any  other  tissue.     Likewise,  although  diphtheria  in  the 
throat  may  spread  in  many  directions,  it  seldom  passes  down  the  esopha- 
gus. 

Some  differences  are  known  to  exist  in  regard  to  local  immunity  as 
observed  in  the  child  and  in  the  adult.  For  example,  ring-worm  of  the 
scalp  is  practically  unknown  among  adults,  whereas  children  under 
seven  years  of  age  are  quite  susceptible  to  the  disease.  These  differences 
may  be  due  to  the  greater  susceptibility  in  general  of  young  tissues  to 
infection,  and  the  local  immunity  constitutes  but  an  index  to  the  gen- 
eral rise  in  resisting  power  accompanying  improvement  in  strength  and 
vitality.  In  some  cases  this  may  be  due  perhaps  to  an  actual  strengthen- 
ing of  local  tissues,  as  in  the  case  of  the  adult  vaginal  mucosa,  which  is 


170  THE    VARIOUS   TYPES   OF   IMMUNITY 

immune  to  the  gonococcus,  whereas  the  thin  and  immature  infantile 
membrane  is  peculiarly  susceptible. 

In  general,  our  knowledge  of  local  immunity  is  quite  incomplete. 
The  subject  is  a  difficult  one,  hence  most  attention  has  been  given  to  the 
study  of  general  immunity.  A  striking  example  of  acquired  local  im- 
munity may  be  seen  in  a  patch  of  psoriasis,  where  the  center  is  observed 
to  be  largely  free  from  scales,  whereas  the  margins  are  quite  active. 

The  question  of  local  immunity  may  be  largely  determined  by  vari- 
ous local  non-specific  factors,  such  as  loss  of  blood  supply  due  to  trau- 
matism,  thrombosis,  tight  bandaging,  etc.,  and  the  action  of  severe 
irritants,  tending  to  produce  necrosis  of  the  tissues. 

4.  The  importance  of  phagocytosis  in  natural  immunity  must  be  em- 
phasized.    Microorganisms    are    constantly    gaining    entrance    to    the 
tissues  through  numerous  small  abrasions  of  the  skin  and  along  the  in- 
testinal and  respiratory  tracts,  and  investigations  have  shown  how  im- 
portant the  wandering  cells  are  in  preventing  infection,  being  ever  on 
guard  and  ready  to  pick  up  and  dispose  of  any  injurious  material.     Even 
after  mild  infection  has  occurred,  the  local  inflammatory  reaction  in 
which  the  phagocyte  is  a  prominent  factor  may  be  so  prompt  in  over- 
coming the  invaders  that  the  host  will  escape  serious  infection. 

The  natural  immunity  of  the  frog  to  anthrax  has  been  shown  to  be 
partly  dependent  upon  the  activity  of  the  leukocytes  in  engulfing  and 
disposing  the  bacilli. 

Similarly  a  mild  irritant  may  produce  hyperemia  and  exudation  or 
local  accumulation  of  leukocytes,  which  aid  in  establishing  a  local  im- 
munity largely  dependent  upon  phagocytosis.  In  this  manner  the 
intraperitoneal  injection  of  sterile  bouillon  or  even  of  salt  solution  may 
produce  exudation  and  increase  the  immunity  to  infection. 

5.  It  may  be  that  even  after  the  introduction  of  a  microorganism  or  its 
toxin  no  harm  results  because  of  a  lack  of  suitable  receptors  on  the  part 
of  the  body -cells  of  the  host  for  union  with  the  pathogenic  agent.     For 
example,  tetanus   toxin,    being    unbound    by   the    cells,  produces   no 
effect  on  the  turtle,  and  antitoxin  is  not  produced.     On  the  other  hand, 
suitable  receptors  may  be  present  that  will  bind  the  toxin,  but  produce 
no  harmful  effects  because  the  body-cells  are  not  susceptible  to  the 
action  of  the  microparasite  or  its  products.     Thus  it  is  asserted  that 
tetanus  toxin  has  no  effect  upon  the  alligator,  although  the  toxin  is 
bound  and  antitoxin  is  produced  by  its  body-cells. 

In  other  instances,  a  host  may  escape  infection  owing  to  the  fact 
that  there  is  a  lowered  affinity  between  a  pathogenic  agent  and  the 


NATURAL   IMMUNITY  171 

body-cells,  so  that  but  a  small  amount  of  harmful  substances  are  bound 
to  the  body-cells,  and  no  particular  harm  results,  whereas  a  larger  dose, 
uniting  with  a  greater  number  of  cells,  is  capable  of  producing  some  dis- 
turbance. 

6.  A  natural  antitoxin  immunity  may  exist,  as  the  immunity  of  the 
alligator  to   tetanus  toxin,   just   mentioned.     Similarly  natural  diph- 
theria antitoxin  may  prevent  infection,   especially  in  those  persons 
known  to  harbor  virulent  bacilli  in  the  fauces.     In  such  instances,  how- 
ever, it  is  difficult  to  exclude  entirely  the  possibility  that  a  previous 
minor  infection  has  occurred,  as  natural  antitoxin  immunity  persists 
much  longer  than  the  passive  immunity  resulting  from  the  administra- 
tion of  an  antitoxin  serum. 

Otto,  who  has  recently  investigated  the  content  of  diphtheria  anti- 
toxin in  the  blood  of  normal  persons,  found  more  than  YW  of  a  unit  of 
antitoxin  in  each  cubic  centimeter  of  the  blood  of  those  who  had  been 
in  close  contact  with  cases  of  diphtheria  without  having  been  sick  them- 
selves; others  usually  had  much  less.  Observations  would  tend  to 
show  that  this  quantity  of  antitoxin  is  generally  sufficient  to  confer 
immunity  to  diphtheria,  and  the  object  of  von  Behring's  method  of 
active  immunization  is  to  induce  the  production  of  at  least  that  much 
antitoxin  by  the  body  itself.  Otto  found  that  diphtheria  carriers,  both 
those  who  had  had  the  disease  and  those  who  had  not,  contained  more 
antitoxin  in  their  blood  than  did  patients  who  had  just  recovered  from 
an  attack.  This  shows  that  the  mere  presence  of  bacilli  in  the  throat 
is  sufficient  to  stimulate  the  production  of  antitoxin,  on  which  the  im- 
munity of  the  carrier  himself  would  seem  to  depend.  Undoubtedly 
physicians  and  nurses  who  are  freely  exposed  to  diphtheria  and  yet 
escape  infection  owe  their  safety  rather  to  an  acquired  immunity  the 
result  of  repeated  contact  with  the  bacilli,  than  to  a  natural  antitoxin 
immunity. 

7.  In  some  instances  a  natural  immunity  may  be  dependent,  at  least 
in  part,  upon  antibacterial  activity,  due  to  the  presence  of  bacteriolysins  and 
bacteriotropins  in  the  body-fluids,  as,  for  example,  that  shown  by  the  dog 
and  the  rat  to  anthrax.     In  other  instances,  however,  a  similar  immunity 
may  be  observed  that  cannot  be  ascribed  to  the  presence  of  antitoxins  or 
bacteriolysins.     In  this  type  of  immunity  microparasites  are  apparently 
unable  to  sustain  themselves,  and  proliferate  in  one  animal,  although 
aggressive  enough  in  another  of  the  same  species. 

8.  An  immunity  to  infection,  especially  with  such  microorganisms  as 
the  anthrax  bacillus,  which  is  markedly  aggressive  and  but  slightly  toxic, 


172  THE   VARIOUS   TYPES   OF  IMMUNITY 

may  be  due  to  the  presence  or  production  of  anti-aggressins.  This  im- 
munity would  seem  to  depend  not  upon  the  bactericidal  properties  of 
the  serum  or  leukocytes  nor  upon  the  antitoxins,  but  on  the  presence  of 
substances  that  prevent  the  microorganisms  from  exercising  their  special 
aggressive  forces. 

9.  Finally,  an  immunity  may  exist  because  the  parasite  or  other  for- 
eign cell  does  not  obtain  suitable  nutrition  in  a  host  and  thus  fails  to  grow. 
This  condition  of^athrepsia,  is  responsible  for  what  has  been  called  athreptic 
immunity.  It  has  been  more  recently  studied  by  Ehrlich,  who  found 
that  upon  transferring  mouse  cancer  to  the  rat,  the  tumor  grew  for  a 
short  time  only,  or  presumably  until  the  special  nutriment  carried  over 
with  the  tumor  was  consumed.  While  there  is  no  experimental  basis 
for  assuming  that  a  similar  condition  may  be  present  in  bacterial  life, 
yet  such  a  cause  may  be  operative  and  should  be  kept  in  mind. 


ACQUIRED  IMMUNITY 

Acquired  immunity  occurs  in  two  distinct  forms:  (1)  Active  and  (2) 
Passive.  A  mixed  form  may  exist,  brought  about  by  a  combination 
of  factors  necessary  for  the  development  of  the  other  two. 

Active  Acquired  Immunity. — Active  acquired  immunity  is  that  form  of 
resistance  to  infection  brought  about  by  the  activity  of  the  cells  of  a  person 
or  animal  as  a  result  of  having  had  the  actual  disease  in  question,  or  as  a 
result  of  artificial  inoculation  with  a  modified  or  attenuated  form  of  the 
causitive  microparasite. 

The  essential  feature  of  this  immunity  is  that  the  cells  and  tissues 
of  persons  or  animals  should,  by  their  own  efforts,  and  as  a  result  of 
their  own  active  struggle  against  the  action  of  a  microparasite  or  its 
products,  overcome  these  and  become  less  susceptible  to  them  than  they 
were  before. 

This  form  of  immunity  is  gained,  therefore,  only  as  the  result  of  an 
active  struggle  between  body-cells  and  infecting  agent,  and  this  battle 
may  be  of  any  degree  of  severity,  ranging  from  an  attack  of  the  disease 
itself  that  may  threaten  life,  down  to  the  most  transitory  and  trivial  re- 
action due  to  the  injection  of  a  minute  dose  of  a  mild  vaccine. 

Active  acquired  immunity  may  be  gained — (1)  By  accidental  infection, 
which  is  the  most  familiar  form  of  acquired  immunity,  and  follows  an 
attack  of  an  infectious  disease,  such  as  scarlet  fever,  measles,  varicella, 
variola,  or  typhus  fever;  (2)  by  inducing  an  attack  of  the  disease  by  arti- 
ficial inoculation.  This  latter  method  of  producing  an  active  acquired 


ACQUIRED    IMMUNITY  173 

immunity  was  illustrated  by  the  ancient,  obsolete,  and  discarded  prac- 
tice of  smallpox  inoculation,  by  which  healthy  persons  were  inoculated 
with  the  virus  of  a  mild  case  of  smallpox,  at  a  time  when  no  epidemics 
existed  and  the  person  was  in  good  general  health  and  able  to  secure 
proper  attention  from  the  outset. 

This  process  of  immunization  is  used  much  more  extensively  in 
veterinary  practice,  where  an  occasional  untoward  or  fatal  result  is  of 
comparatively  little  importance  if  by  its  means  an  outbreak  can  be  con- 
trolled or  the  great  majority  of  the  animals  saved.  As  a  rule,  an  at- 
tempt is  made  to  render  the  induced  disease  as  mild  as  possible  by — (a) 
Using  a  small  amount  of  infective  material;  (6)  by  inoculating  it  through 
an  unusual  avenue;  or  (c)  by  inoculating  it  at  a  time  when  the  animals 
are  naturally  less  susceptible,  or  (d)  by  a  combination  of  these  methods. 
For  example,  Texas  cattle  fever,  which  is  due  to  a  protozoan  (Piro- 
plasma  bigeminum)  conveyed  by  the  bites  of  infected  ticks,  may  be 
combated  by  exposing  calves  while  still  milk  fed  to  the  bites  of  a  few 
infected  ticks.  Another  method  consists  in  injecting  a  small  amount  of 
blood  from  an  infected  animal  directly  into  the  jugular  vein.  The  ob- 
ject is  to  induce  a  mild  attack  of  the  disease.  Occasionally  a  severe  or 
fatal  reaction  occurs,  but  the  number  of  these  untoward  results  is  much 
lower  than  the  mortality  among  untreated'  animals. 

(3)  Active  immunity  may  also  be  gained  by  vaccination,  i.  e.,  by  in- 
oculation with  a  virus  or  microparasite  or  its  products,  modified  and 
attenuated  by  passage  through  a  lower  animal  (Jennerian  vaccination) 
or  by  various  other  means,  as  age,  unfavorable  cultural  conditions,  heat, 
germicides,  etc.  (Pasteurian  vaccination  or  bacterination) .  These  subj ects 
are  considered  more  fully  in  the  chapter  on  Active  Immunization. 

Active  immunity,  whether  induced  accidentally  or  artificially,  may 
be  antitoxic,  as  after  recovery  from  diphtheria  or  as  the  result  of  active 
immunization  with  diphtheria  toxin,  as  by  von  Behring's  method;  or 
antibacterial,  as  the  immunity  following  typhoid  fever  or  induced  by 
typhoid  vaccination,  and  largely  dependent  upon  the  presence  of  bac- 
teriolysins  in  the  circulating  fluids. 

During  the  process  of  active  immunization  an  animal  not  infre- 
quently fails  to  react  to  relatively  large  doses  of  toxin,  and  at  the  same 
time  the  quantity  of  antibody  in  the  body-fluid  may  decrease.  This 
phenomenon  has  been  explained  as  being  due  to  atrophy  of  the  receptors 
of  the  body-cells  (receptoric  atrophy),  whereby  the  toxin  fails  to  exert 
its  deleterious  influence  because  it  fails  to  unite  with  the  body-cells. 
It  is  curious,  however,  that  the  toxin  is  innocuous  when  present  in  a 


1 74  THE   VARIOUS   TYPES   OF   IMMUNITY 

free  state  within  the  body-fluids,  even  though  unbound  to  the  body- 
cells;  this  condition  is  not  well  understood,  and  may  be  dependent  upon 
other  factors.  A  rest  may  restore  the  activity  of  the  receptors  'and  cells, 
a  fact  that  is  well  recognized  in  the  immunization  of  horses  for  the  prepara- 
tion of  antitoxin.  Not  infrequently  a  rabbit  fails  to  produce  hemolytic 
amboceptor  if  the  injections  of  erythrocytes  are  too  frequent.  After 
a  rest,  however,  the  animal  may  react  promptly  with  much  smaller 
doses. 

Passive  Acquired  Immunity. — As  the  name  indicates,  this  is  a  form 
of  immunity  that  depends  upon  defensive  factors  not  originating  in  the 
person  or  animal  protected,  but  is  passively  acquired  by  the  injection  of 
serum  from  one  that  has  acquired  an  active  immunity  to  the  disease  in 
question. 

This  is  a  sort  of  secondary  immunity,  acquired  by  virtue  of  having 
received  antibodies  actively  formed  by  another  animal  that  has  had  to 
resist  the  infecting  agent  in  order  to  produce  them.  Two  well-known 
examples  of  this  type  of  serums  are  the  diphtheria  and  tetanus  anti- 
toxins. These  are  produced  by  injecting  horses  with  successive  doses 
of  the  respective  toxins.  The  horses  are  required  to  combat  the  effects 
of  the  toxins,  and  acquire  an  active  immunity  of  increasing  grade,  due  to 
the  production  of  antitoxins.  When  the  animals  are  bled,  the  antitoxin- 
laden  serum,  separated  from  the  corpuscular  elements,  may  be  used  for 
conferring  an  immunity  in  a  person  or  another  animal  simply  by  injecting 
the  serum,  the  latter  receiving  and  enjoying  an  immunity  in  a  passive 
manner. 

Passive  immunity  is  specific,  that  is,  the  serum  of  an  animal  im- 
munized against  one  microorganism  will  protect  a  second  animal  against 
that  and  against  no  other.  This  type  of  immunity  is  acquired  just  as 
soon  as  the  immune  serum  has  become  mixed  with  the  blood  of  the  per- 
son or  animal  injected,  and  there  is  no  negative  phase.  Hence  in 
severe  infections  our  hopes  of  specific  therapy  rest  on  the  production  of 
passive  immunity.  No  matter  how  sick  the  recipient  may  be,  under 
ordinary  circumstances  the  immune  serum  produces  no  further  dis- 
turbance than  would  be  expected  from  the  injection  of  a  normal  serum. 
The  recipient's  body-cells  have  no  additional  burdens,  or  very  slight 
ones  only,  to  bear  and  these  are  more  than  counterbalanced  by  the  re- 
lease from  combat  with  toxic  substances  neutralized  by  the  antibodies 
in  the  immune  serum.  Unfortunately,  this  field  of  therapy  is  limited, 
although  recent  discoveries  are  indicating  the  reasons  for  failure,  and 
when  these  are  eliminated,  the  field  of  usefulness  will  be  much  extended. 


ACQUIRED    IMMUNITY  175 

Passive  immunity  is  of  shorter  duration  than  active  immunity,  and 
the  former  is  especially  indicated  in  prophylaxis  for  warding  off  an 
acute  infection  that  has  a  relatively  short  incubation  period.  The  de- 
gree of  passive  immunity  is  also  seldom  equal  to  that  of  an  active  im- 
munity. The  antibodies  produced  by  our  own  cells  are  more  lasting 
and  possess  higher  protective  value.  This  is  an  important  factor  in 
von  Behring's  method  of  immunization  in  diphtheria,  when  a  small 
amount  of  toxin  loosely  bound  to  antitoxin  is  injected  in  the  belief  that 
the  toxin  becomes  dissociated  and  serves  to  stimulate  our  body-cells  into 
producing  our  own  antitoxin. 

Passive  acquired  immunity  is  usually  antitoxic,  as,  for  example, 
that  induced  by  the  administration  to  man  of  diphtheria  antitoxin 
prepared  by  the  body-cells  of  the  horse.  Antibacterial  serums  may 
likewise  induce  a  passive  immunity,  as,  for  instance,  that  used  in  im- 
munization against  plague. 

It  is  evident,  therefore,  that  the  processes  whereby  infections  are 
overcome  and  immunity  is  conferred,  and  the  general  reactions  that 
follow  the  introduction  into  the  body  of  modified  antigens  in  the  prac- 
tice of  immunization,  are  complex  processes,  and  in  none  is  one  anti- 
body produced  or  solely  responsible  for  the  resulting  immunity.  The 
properties  and  action  of  the  known  antibodies  are  considered  in  sub- 
sequent chapters,  particular  attention  being  given  to  methods  for  de- 
termining their  presence  in  the  body-fluids,  which  serve  as  an  aid  to  the 
diagnosis  of  infection  as  based  upon  the  general  law  that  the  antibody 
is  specific  for  its  antigen,  and  so,  when  the  presence  of  an  antibody 
is  demonstrated,  it  may  be  assumed  that  the  antigen  is  or  has  been 
present. 

Nothing  is  known  concerning  the  nature  of  the  immunity  that  is  ac- 
quired against  several  infections,  such  as  scarlet  fever,  measles,  small- 
pox, etc.,  nor  will  much  be  known  until  the  causes  that  give  rise  to  these 
conditions  have  been  discovered. 

Theory  of  Vaughan. — According  to  Vaughan,  the  inability  of  a  bac- 
terial cell  to  grow  in  the  animal  body  either  because  it  cannot  feed  upon 
the  protein  of  the  body  or  because  it  is  itself  destroyed  by  the  ferments 
elaborated  by  the  body-cells  explains  all  forms  of  bacterial  immunity, 
either  natural  or  acquired.  Thus  in  antitoxin  immunity  the  toxin  is 
regarded  as  a  ferment  that  splits  up  the  proteins  of  the  body-cells,  setting 
the  protein  poison  free.  The  body-cells  react  with  the  formation  of  an 
antiferment  or  antitoxin,  which  neutralizes  the  toxin  and  prevents 
cleavage.  The  toxin  itself  is  regarded  as  harmful  only  in  so  far  as  it  is 


176 


THE   VARIOUS   TYPES   OF  IMMUNITY 


able  to  set  free  the  protein  poison  responsible  for  the  symptoms  of  the 
infection. 

Natural  immunity  to  any  infection,  according  to  Vaughan's  theory,  is 
explained  as  being  due  to  an  inability  of  the  infecting  agent  to  grow  in 
the  animal  body. 

Acquired  immunity,  due  to  recovery  from  an  infection  or  occurring  as 
a  result  of  vaccination,  is  regarded  as  the  outcome  of  the  development 
in  the  body,  during  the  course  of  the  infective  process,  of  a  specific  fer- 
ment that,  on  renewed  exposure,  immediately  destroys  the  infection. 
The  vaccine  is  the  same  protein  that  causes  the  disease,  so  modified  that 
it  will  not  produce  the  disease,  but  yet  so  little  altered  that  it  will  stim- 
ulate the  body-cells  to  form  a  specific  ferment  that  will  promptly  and 
quickly  destroy  the  infecting  agent  on  exposure. 

The  various  kinds  of  immunity  and  the  factors  probably  concerned 
in  their  production,  may  be  summarized  as  follows : 

1.  Due  to  non-specific  factors: 

1.  Barrier  of  epithelium. 

2.  Various  secretions. 

3.  A  particular  route  of  infection  may  be  nec- 

essary, aided  by  the  biologic  nature  of 
the  invading  bacterium. 


Natural  Immunity 


2.  Due  to  local  tissue  immunity  and  selective  ac- 

tion of  micro-parasites  for  certain  tissues. 

3.  Due  to  phagocytosis. 

4.  Due  to  lack  of  suitable  receptors  of  body-cells 

for  a  particular  bacterium. 

5.  Due  to  natural  antitoxins. 

6.  Due  to  natural  bacteriolysins. 

7.  Due  to  anti-aggressins. 

8.  Due  to  lack  of  suitable  food  material — athrep- 

sia. 


Acquired  Immunity  1 


I 


Active  (accidentally  or  |  1.  Antitoxic, 
artificially  acquired)  J  2.  Antibacterial. 

1.  Antitoxic. 

2.  Antibacterial. 


Passive 


CHAPTER  X 
PHAGOCYTOSIS 

Historic. — As  Lord  Lister  stated  in  1896,  "If  ever  there  was  a 
romantic  chapter  in  pathology,  it  has  surely  been  that  of  the  story  of 
phagocytosis."  The  author  of  this  "story"  is  Elie  Metchnikoff.  His 
early  researches .  on  phagocytosis  in  the  lowly  organized  forms  of  life 
constituted  the  starting-point  for  an  entirely  new  series  of  researches  on 
the  subject  of  Immunity,  and  his  treatise  on  the  "Comparative  Pathology 
of  Inflammation"  must  ever  remain  a  most  entertaining  work  and  a 
medical  classic. 

Several  observers  before  Metchnikoff  had  considered  that  leuko- 
cytes might  assist  in  bringing  about  the  destruction  of  microparasites. 
Panum  (1874)  suggested  that  the  bacilli  of  putrefaction  might,  by  mak- 
ing their  way  into  the  blood-corpuscles  and  being  carried  off  to  the 
lymph-glands,  spleen,  etc.,  thus  disappear  from  the  body-fluids.  Carl 
Roser  (1881)  had  also  observed  the  ability  of  certain  "contractile  cells" 
to  ingest  the  enemy  that  enters  the  animal  body.  These  statements, 
however,  were  poorly  supported  by  scientific  data,  and  the  subject  was 
>not  followed  in  subsequent  research.  As  the  result  of  zoologic  studies, 
Metchnikoff  was  led  to  discover  the  part  played  by  body-cells  in  the 
processes  of  immunity.  He  observed  that  when  a  food-particle  arrives  in 
the  vicinity  of  a  simple  unicellular  organism  as  an  ameba,  the  latter, 
by  reason  of  its  "irritability, "  moves  forward  and  sends  out  processes  of 
its  protoplasm  (pseudopodia)  that  flow  around  the  particle  and  finally 
gather  it  into  the  interior  of  the  cell.  The  particle  then  undergoes  a 
process  of  intracellular  digestion,  losing  its  sharp  outline  and  clear  ap- 
pearance, becoming  granular,  and  disappearing  within  the  protoplasm 
of  its  host.  Similar  studies  were  made  of  the  processes  of  nutrition  in 
many  unicellular  animals,  then  through  actininas,  sponges,  and  similar 
animals  of  transparent  and  simple  organization. 

Metchnikoff  was  primarily  a  biologist,  and  up  to  this  time  was 
mainly  interested  in  the  processes  of  nutrition  hi  the  simple  forms  of 
animal  fife.  At  this  stage,  however,  he  became  greatly  impressed  by 
Cohnheim's  description  of  the  phenomena  of  inflammation.  The  dia- 
pedesis  of  leukocytes  through  the  walls  of  the  blood-vessels  in  an 
12  177 


178  PHAGOCYTOSIS 

inflammatory  reaction  impressed  him  as  significant  and  similar  to  the 
movements  of  the  ameba  in  its  process  of  feeding,  and  led  to  com- 
parative studies  in  inflammation  in  the  lower  forms  of  life,  where 
processes  were  much  simpler  to  watch  and  may  indicate  what  occurs 
in  the  higher  vertebrates. 

He  found  that  when  daphnias  are  invaded  by  the  spores  of  a  yeast, — 
the  monospora, — they  may  multiply  in  the  body  of  the  host  and  bring 
about  its  destruction.  When,  however,  a  few  spores  gained  access,  he 
found  that  the  daphnia's  leukocytes  approached  them,  formed  a  wall 
around  them,  and  finally  brought  about  their  destruction  by  a  process 
of  digestion.  He  also  observed  that  if  rose  prickles  were  stuck  into 
large  bipinnaria  larvae  of  star  fish,  these  were  soon  enveloped  in  a  mass 
of  ameboid  cells.  From  this  he  concluded  that,  in  inflammation,  the 
exudation  of  leukocytes  may  be  regarded  as  a  reaction  against  any  sort 
of  injury,  whether  mechanical  or  due  to  bacterial  invasion. 

Metchnikoff  traced  this  defensive  reaction  against  an  invading 
microorganism  from  small  invertebrates  up  to  man,  and  showed  that 
instead  of  bacteria  attacking  leukocytes  and  forcing  a  passage  into  them, 
as  was  then  believed,  they  were  indeed  pursued  and  engulfed  by  the 
leukocytes.  Connecting  his  various  discoveries,  he  was  able  to  formu- 
late the  idea  that  "the  intracellular  digestion  of  unicellular  organisms 
and  of  many  invertebrates  had  been  hereditarily  transmitted  to  the 
higher  animals,  and  retained  in  them  by  the  ameboid  cells  of  mesodermic 
origin.  These  cells,  being  capable  of  ingesting  and  digesting  all  kinds* 
of  histologic  elements,  may  apply  the  same  power  to  the  destruction  of 
microorganisms."  To  a  cell,  and  especially  to  a  leukocyte,  possessing 
this  activity  and  power  he  applied  the  name  phagocyte,  because  of  its 
ability  to  act  as  a  scavenger,  gathering  up  the  living  and  dead  material. 


THE  ORIGINAL  THEORY  OF  PHAGOCYTOSIS 

The  theory  as  originally  adduced  by  Metchnikoff  regarded  the  leu- 
kocytes and  certain  other  cells  as  specific  fighting  cells,  able  to  engulf 
and  consume  living  as  well  as  dead  bacteria  and  cellular  debris.  The 
outcome  of  any  infection  would  depend  upon  the  success  or  failure  of  the 
phagocytes  to  overcome  the  invaders:  if  they  were  successful  in  in- 
gesting all  the  bacteria,  their  victory  meant  recovery;  if,  on  the  other 
hand,  they  were  destroyed  by  the  invaders,  the  host  was  overcome  by 
the  infection. 

Phagocytes  may  ingest  not  only  living  and  dead  bacteria,  but  also 


FIG.  44. — PHAGOCYTOSIS — MACROPHAGES. 

A  smear  of  peritoneal  exudate  from  a  guinea-pig  twenty-four  hours  after  injec- 
tion with  3  c.c.  of  a  5  per  cent,  suspension  of  pigeon's  blood.  Note  that  the  corpuscles 
(nucleated)  are  being  ingested  by  the  large  endothelial  cells  of  the  peritoneum. 


PIG.  45 . — PHAGOCYTOSIS — MACROPHAGES. 

A  smear  of  peritoneal  exudate  from  the  same  guinea-pig  forty-eight  hours  after 
injection  with  pigeon's  blood.  Note  the  large  numbers  of  corpuscles  ingested  by  the 
endothelial  cells.  In  most  instances  the  corpuscles  have  undergone  digestion,  the 
nuclei  being  more  resistant.  These  nuclei  are  shrunken,  and  in  some  instances  are 
broken  up.  The  extracellular  red  blood-corpuscles  are  swollen  and  stain  lightly. 


VARIETIES    OF    PHAGOCYTES  179 

red  corpuscles,  cellular  debris,  inorganic  particles,  such  as  coal-dust,  and 
even  soluble  substances,  such  as  bacterial  toxins. 

Subsequent  discoveries  have  shown  that  many  other  factors  are 
present  that  considerably  modify  the  workings  of  so  simple  a  process. 
Metchnikoff  has,  therefore,  modified  his  theory  from  time  to  time  as 
new  discoveries  were  made,  but  has  always  preserved  the  primary  im- 
portance of  the  phagocyte  itself. 

Before  stating  the  revised  theory  as  it  stands  today,  we  will  describe 
the  kind  of  cells  that  may  act  as  phagocytes,  and  consider  the  methods 
and  reasons  why  these  cells  assume  the  functions  of  phagocytes. 

VARIETIES  OF  PHAGOCYTES 

Not  only  leukocytes,  but  other  body-cells,  have  been  found  active 
in  the  processes  of  phagocytosis.  Metchnikoff  has  divided  the  phago- 
cytes into  two  great  classes : 

1.  Microphages — principally  the  polynuclear  neutrophilic  leukocytes. 
(See  Fig.  46.)    The  eosinophilic  leukocytes  are  also  included  in  this  group, 
but  are  of  doubtful  importance  and  weak  in  phagocytic  powers.     The 
small  lymphocyte  may  be  included  in  this  class,  although  it  is  usually  con- 
sidered with  the  second  group.     0 

2.  Macrophages,    principally    the    large    mononucleaf    leukocytes; 
ameboid  cells  of  the  spleen  and  lymphatic  glands;    alveolar  cells  of  the 
lung;    endothelial  cells  of  the  serous  cavities  and  lymph-spaces;    bone- 
corpuscles  and  giant-cells  of  bone-marrow  and  embryonic  connective- 
tissue  cells  (Figs.  44  and  45).    As  shown  experimentally  by  Rous  and 
Jones  the  phagocytic  powers  of  fibroblasts  appear  to  be  very  feeble.1 

The  most  important  are  the  leukocytes,  especially  the  polynuelear 
leukocytes  and  the  large  lymphocytes  of  the  blood.  All  the  leukocytes, 
however,  have  phagocytic  powers,  as  is  well  seen  in  opsonic  determina- 
tions. Eosinophiles  are  seldom  known  to  ingest  bacteria,  but  in  in- 
fections with  animal  parasites,  or  after  the  injection  of  extracts  of  ani- 
mal parasites,  both  a  local  and  a  general  increase  in  eosinophilous  forms 
may  be  observed. 

Small  lymphocytes  are  much  less  active  than  the  large,  presumably 
because  they  contain  less  of  the  mobile  cytoplasm,  and  consist  chiefly 
of  the  structurally  fixed  nuclear  substance,  and  while  they  take  up  but 
a  small  number  of  bacteria,  they  may  be  observed  to  contain  Various 
other  cells,  such  as  red  corpuscles  and  cellular  debris. 

Besides  the  leukocytes,  some  of  the  tissue-cells  which  are  free  01 
1  Jour.  Exper.  Med.,  1917,  25,  189. 


180  PHAGOCYTOSIS 

have  the  power  of  becoming  so  are  actively  phagocytic.  Endothelial 
cells  of  the  lymph-spaces  and  serous  cavities  are  especially  active,  not 
only  in  the  phagocytosis  of  other  cells  and  cellular  debris,  but  also  of 
various  bacteria.  In  exceptional  instances  epithelial  cells  may  act  as 
phagocytes.  In  the  presence  of  an  irritant  these  cells  may  become  de- 
tached and  act  as  phagocytes;  this  is  exemplified  in  the  case  of  chronic 
passive  congestion  of  the  lungs,  in  which  the  alveolar  cells  of  the  lung 
ingest  the  hemosiderin  formed  and  deposited  (heart  failure  cells). 

Relation  of  the  Cell  Types  to  Infection.— The  kind  of  cells  that  take 
part  in  phagocytosis  is  determined  to  some  extent  by  the  nature  of  the 
irritant.  Thus  in  acute  pyogenic  infections  the  polynuclear  cell  is 
found  to  be  most  active.  (See  Fig.  46.)  It  is  extremely  rare  to  find  these 
cells  containing  bacilli  in  the  tissues,  although  they  will  take  them  up 
readily  enough  under  the  artificial  conditions  of  an  opsonic  determina- 
tion. (See  Fig.  53.)  In  chronic  bacterial  infection,  such  as  tubercu- 
losis and  syphilis,  and  in  infections  with  various  fungi,  the  small  lympho- 
cyte and  macrophages  are  the  types  most  concerned. 

Experimental  evidence  regarding  lymphocytic  activity  is  quite  con- 
tradictory. Undoubtedly  many  of  the  cells  in  the  lymphocytic  accumu- 
lations seen  hi  such  conditions  as  tuberculosis  and  syphilis  are  not  really 
lymphocytes  from  the  blood,  but  are  newly  formed  cells  of  the  tissues. 
There  is  no  direct  means  of  inducing  experimentally  a  local  accumula- 
tion of  lymphocytes  similar  to  that  induced  by  most  any  irritant, 
resulting  in  an  outpouring  of  polynuclear  cells.  Long-continued  in- 
toxication of  animals  may  result  in  increasing  the  numbers  of  lympho- 
cytes, but  the  local  introduction  of  the  toxin  leads  to  an  accumulation 
of  polynuclear  cells,  rather  than  lymphocytes.  Reckzeli 1  found  that 
in  lymphatic  leukemia,  in  which  the  lymphocytes  greatly  exceed  the 
polynuclears,  the  pus  from  an  acute  lesion  or  the  fluid  from  the  vesicles 
produced  by  cantharides,  contained  practically  no  lymphocytes,  but 
was  composed  of  the  usual  polynuclear  cell  forms.  Wlassow  and  Sepp 2 
state  that  lymphocytes  are  not  capable  of  ameboid  movement  or 
phagocytosis  at  ordinary  body  temperature;  Wolff,3  on  the  other  hand, 
claims  that  tetanus  and  diphtheria  toxins  produce  lymphocytosis  in 
experimental  animals,  and  Zieler4  claims  that  in  the  skin  of  rabbits 
exposed  to  the  Finsen  light  active  migration  of  lymphocytes  takes  place 
during  the  reaction.  General  lymphocytosis  may  be  produced  experi- 
mentally by  the  injection  of  pilocarpin  and  muscarin,  but  these  bear  no 
relation  to  the  vital  process  of  phagocytosis,  as  they  are  apparently  ex- 

1  Zeit.  f.  klin.  Med.,  1903,  50,  51.          2  Virchow's  Archives,  1904,  176,  185. 
a  Berl.  klin.  Woch.,  1904,  41,  1273.        4  Central.!.  Pathol.,  1907,  18,  289. 


FIG.  46. — POSITIVE  CHEMOTAXIS.     PHAGOCYTOSIS  OF  STAPHYLOCOCCI. 


CHEMOTAXIS  181 

truded  from  the  lymphoid  organs  by  contraction  of  the  smooth  muscles 
(Harvey1). 

As  previously  stated,  the  eosinophiles  undergo  a  marked  increase 
during  infections  with  various  animal  parasites. 

The  typical  macrophages,  such  as  the  endothelial  cells  of  serous 
cavities  (Figs.  44  and  45)  and  the  lymph-spaces,  are  mostly  concerned  in 
the  phagocytosis  of  other  cells  and  inorganic  material.  Erodie2  con- 
siders the  phagocytosis  of  leukocytes  and  red  corpuscles  by  the  endo- 
thelial cells  of  the  lymph-glands  and  the  spleen  as  the  normal  end  of 
these  cells.  It  is  a  mistake  to  believe,  however,  that  they  do  not  in- 
gest bacteria,  since  endothelial  cells  are  extremely  active  phagocytically 
for  bacteria,  and,  on  the  other  hand,  polynuclear  leukocytes  may  be 
observed  to  contain  red  corpuscles,  especially  when  aided  by  a  suitable 
opsonin. 

CHEMOTAXIS 

An  important  question  in  the  study  of  the  phenomena  of  phago- 
cytosis is  the  manner  in  which  the  various  leukocytes  and  other  body- 
cells  are  attracted  to  a  focus  of  infection  and  brought  into  contact  with 
the  microparasites  or  other  foreign  substances.  It  must  be  assumed 
that  some  means  of  communication  must  exist  between  this  point  and 
the  leukocytes  in  the  circulating  blood.  Since  there  is  no  direct  com- 
munication by  way  of  the  nervous  system  or  other  structural  route,  it 
would  appear  that  the  only  mode  of  communication  is  through  the  body- 
fluids.  Chemical  agencies,  produced  either  directly  by  the  bacteria  or  other 
foreign  substance,  or  indirectly  by  their  action  upon  cells  at  the  site  of  resi- 
dence in  the  tissues,  are  regarded  as  furnishing  the  attractive  forces  that  are 
transmitted  through  the  body-fluids  and  exert  what  has  been  called  chemo- 
taxis. 

The  movement  of  a  cell  in  response  to  a  chemical  stimulus  is  a  phe- 
nomenon that  is  displayed  by  almost  all  motile  and  unicellular  organ- 
isms, whether  animal  or  vegetable,  and  by  the  leukocytes  and  other  un- 
fixed cells  of  the  higher  animals.  As  a  rule,  chemical  stimuli  serve  to 
attract  cells  to  the  site  of  infection,  thus  constituting  what  is  known  as 
positive  chemotaxis;  on  the  other  hand,  the  stimuli  may  fail  to  attract 
or  actually  repel  the  cells,  or  be  so  powerful  as  to  paralyze  thorn  en 
route,  this  constituting  negative  chemotaxis. 

Positive  Chemotaxis. — That  leukocytes  reach  the  site  of  an  infec- 
tion because  of  chemical  substances  produced  by  bacteria  at  this  point 

1  Jour.  Physiol.,  1906,  35,  115.         2  Jour.  Anat.  and  Physiol.,  1901,  35,  142. 


182  PHAGOCYTOSIS 

was  first  clearly  demonstrated  by  Leber1  in  1879.  This  writer  ob- 
served that  in  keratitis  leukocytes  invaded  the  avascular  cornea 
from  the  distant  vessels,  not  in  an  irregular  manner,  but  direct  to  the 
point  of  infection,  where  they  accumulated.  As  dead  cultures  of 
staphylococci  produced  a  similar  although  a  less  pronounced  accumu- 
lation of  leukocytes,  Leber  sought  the  chemotactic  substance  in  their 
bodiesrand  isolated  a  crystalline,  heat-resisting  substance — phogosin — 
which  attracted  leukocytes  in  the  tissues. 

Since  these  fundamental  studies  were  made  many  other  investiga- 
tions, with  various  chemical  substances  of  many  different  origins,  have 
been  undertaken  upon  leukocytes,  amebse,  ciliata,  and  plasmodial  forms, 
indicating  that  chemical  substances  are  mainly  concerned  in  exerting 
either  a  positive  or  a  negative  chemotactic  influence. 

Experimental  evidence  tends  to  show  that  cells  respond  to  stimuli 
of  various  kinds  chiefly  through  the  effect  of  these  stimuli  upon  surface 
tension:  if  they  decrease  the  surface  tension,  the  cell  goes  forward;  if 
they  increase  the  tension,  the  cell  recedes. 

The  behavior  of  leukocytes  in  inflammation  may  be  explained  on 
these  purely  physical  grounds.  At  the  site  of  cell  injury  or  infection 
chemical  substances  are  produced  that  tend  to  lower  the  surface  tension 
of  leukocytes  and  thus  exert  a  positive  chemotactic  influence.  These 
chemical  stimuli  are  transmitted  by  the  body-fluids  to  the  nearest  cap- 
illaries, where  they  enter  through  the  vessel-wall  and  come  in  relation 
with  the  slowly  moving  peripheral  leukocytes.  The  leukocytes  will  be 
brought  into  touch  by  the  chemotactic  substances  most  largely  on  the 
side  from  which  the  substances  diffuse ;  accordingly,  the  surface  tension 
being  least  nearest  the  stomata  in  the  capillary  wall,  this  results  in  the 
formation  of  pseudopodia,  and  motion  in  this  direction,  dragging  the 
nucleus  along  in  an  apparently  passive  manner.  Those  cells,  therefore, 
containing  most  of  the  mobile  cytoplasm,  such  as  the  polynuclear  leuko- 
cytes, are  chiefly  affected  in  these  processes;  those  containing  little 
cytoplasm  and  a  relatively  large  and  dense  nucleus,  such  as  the  lympho- 
cytes, are  affected  primarily  to  a  much  less  extent. 

Once  outside  of  the  vessel-wall,  the  leukocytes  tend  to  move  toward 
the  focus  from  which  the  chemotactic  substance  comes.  If  the  leuko- 
cyte meets  a  substance  that  greatly  lowers  its  surface  tension,  it  will  flow 
around  the  object  and  inclose  it,  this  constituting  phagocytosis.  The 
toxins  of  the  ingested  bacteria  may  kill  the  cell,  or  so  equalize  surface 
tension  that  movement  ceases.  Otherwise  the  leukocytes  tend  to  move 
1  Fortschritte  der  Med.,  1888,  6,  460. 


CHEMOTAXIS  183 

forward  until  checked  by  any  one  of  several  influences,  as  pointed  out 
by  Wells, — as  (1)  Until  the  chemotactic  substance  has  been  used  up  or 
removed,  or  from  any  of  the  causes  that  terminate  inflammation;  (2) 
the  leukocytes  may  reach  a  point  where  the  chemical  stimulant  is  so 
generally  diffused  that  surface  tension  is  decreased  equally  in  all  di- 
rections and  motility  stops;  (3)  the  leukocytes  may  reach  a  place  where 
toxins  or  other  chemical  substances  coagulate  their  cytoplasm  or  fer- 
ments cause  their  solution;  (4)  they  may  be  blocked  by  a  dense  wall  of 
leukocytes  and  other  cells  while  being  held  fixed  by  the  chemical  attrac- 
tion that  diffuses  through  this  wall.  These  factors  would  explain  the 
formation  of  the  wall  of  leukocytes  about  an  area  of  infection.  When, 
for  example,  the  abscess  has  ruptured  or  has  been  incised,  with  removal 
of  the  chemotactic  substances,  there  may  be  less  chemotactic  substances 
in  the  center  of  the  inflamed  area  than  there  is  further  out;  hence  the 
leukocytes  will  move  away  from  the  center  toward  the  periphery,  follow- 
ing the  chemotactic  substances  back  into  the  blood-vessel  and  lymph- 
stream.  This  would  explain  the  dispersion  of  living  leukocytes  at  the 
close  of  an  inflammatory  process. 

General  leukocytosis  can  be  explained  equally  well  by  assuming  that 
the  chemotactic  substances  from  the  area  of  inflammation,  reaching  the 
blood-stream,  pass  through  the  bone-marrow,  lowering  surface  tension 
and  attracting  leukocytes  into  the  blood-stream  as  long  as  it  contains 
more  chemotactic  substances  than  the  marrow. 

The  exact  chemical  nature  of  chemotactic  substances  is  unknown. 
In  bacterial  infection  the  toxins,  and  especially  the  protein  of  dead  micro- 
organisms, are  regarded  as  mainly  responsible  for  the  occurrence  of  posi- 
tive chemotaxis.  Chemotaxis  and  phagocytosis  of  chemically  inert 
particles,  such  as  coal-dust,  stone-dust,  and  pigments,  are  more  difficult 
to  explain  on  this  physical  basis  of  alteration  in  surface  tension.  It  is 
probable  that  the  death  of  tissue-cells,  brought  about  by  these  materials, 
may  produce  the  chemical  stimulant  responsible  for  a  mild  but  definite 
chemotactic  influence.  Although  the  movement  of  amebse  and  similar 
fogtier  animals  cannot  be  fully  explained  on  this  physical  basis,  the 
surface  tension  theory  best  explains  leukocytic  movement.  Although 
the  ameba  may  possess  some  special  property  that  endows  it  with  the 
power  of  selecting  and  engulfing  a  food  particle,  it  would  appear  to  be 
entirely  unreasonable  to  assume  that  a  simple,  undifferentiated,  and 
naked  leukocyte  possesses  similar  powers.  The  physical  theory,  there- 
fore, appears  to  be  the  most  reasonable  offered  in  explanation  of  the 
ameboid  movements  of  these  simple  cells. 


184  PHAGOCYTOSIS 

Negative  Chemotaxis. — In  nearly  all  infections  we  find  that  leuko- 
cytes are  attracted  in  large  numbers  into  the  involved  area,  i.  e.,  nearly 
all  bacteria  give  off  substances  that  are  positively  chemotactic.  In  cer- 
tain infections,  however,  we  may  find  the  tissues  poor  in  leukocytes,  as 
exemplified  in  infections  due  to  the  presence  of  virulent  streptococci. 
This  negative  chemotaxis  is  more  difficult  of  explanation.  Kantlack 
doubts  the  existence  of  really  negative  chemotactic  action  upon  leuko- 
cytes. Verigo1.  also  considers  that  as  yet  no  actual  negative  chemo- 
tactic substances  have  been  satisfactorily  demonstrated;  certainly  no 
marked  example  o£  negative  chemotaxis  has  been  shown  since  methods 
involving  the  study  of  phagocytosis  in  vitro  have  been  devised.  It  is 
true  that  virulent  bacteria  appear  to  repel  the  leukocytes,  but,  as 
Kantlack  has  pointed  out,  these  are  not  necessarily  examples  of  nega- 
tive chemotaxis,  and  it  is  probable  that  the  paucity  in  numbers  of  the 
leukocytes  about  such  an  area  of  inflammation  is  due  to  their  over- 
stimulation  or  paralysis  and  destruction  of  the  powerful  ferments  that 
are  given  off  by  the  bacteria.  Thus  Metchnikoff  has  asserted  that 
leukocytes  might,  after  a  time,  be  attracted  toward  substances  that 
would  kill  them.  Therefore,  while  leukocytes  will  migrate  freely 
toward  substances  that  would  kill  them,  they  may  be  destroyed  before 
they  reach  the  inflammatory  area,  or,  having  reached  there,  are  promptly 
destroyed  and  pass  into  solution  (Fig.  47). 

While  it  is  doubtful,  therefore,  whether  substances  are  produced  by 
bacteria  that  actually  repel  leukocytes,  the  point  has  not  been  definitely 
settled.  If  such  substances  exist,  it  would  appear  that  they  are  closely 
identified  with  either  the  endotoxins  or  the  aggressins,  the  latter  being 
definite  secretory  products  of  bacteria  that  neutralize  opsonins  and 
retard  phagocytosis.  In  many  instances  it  is  probable  that  the  same 
substances  that  exert  a  positive  chemotaxis  are,  when  concentrated, 
negatively  chemotactic,  through  overstimulation  and  paralysis  of  the 
leukocytes.  With  diminution  in  the  numbers  or  vitality  of  bacteria 
and  dilution  of  their  chemotactic  substances,  this  inhibiting  influence  is 
removed  and  the  leukocytes  are  attracted  to  the  focus  of  infection,  thus 
explaining  in  a  way  those  instances  in  which  positive  chemotaxis  is 
observed  to  follow  a  primary  period  of  negative  chemotaxis. 

RESULTS  OF  PHAGOCYTOSIS 

After  phagocytosis  has  been  accomplished,  the  fate  of  the  engulfed 
objects  depends  upon  their  nature.     In  general  they  undergo  a  process 
1  Arch.  d.  med.  Exper.,  1901,  13,  585. 


FIG.  47. — NEGATIVE  CHEMOTAXIS. 

A  smear  of  exudate  from  the  peritoneal  cavity  of  a  guinea-pig  twenty-four 
hours  after  injection  with  virulent  streptococci.  The  exudate  was  thin,  serous,  and 
tinged  with  hemoglobin.  Note  the  large  numbers  of  streptococci  and  relatively  few 
leukocytes. 


RESULTS    OF    PHAGOCYTOSIS  185 

of  digestion.  The  ameba,  for  example,  is  able  to  kill  and  digest  en- 
gulfed material  through  an  intracellular  ferment  regarded  as  a  form  of 
trypsin,  demonstrated  by  Mouton1  and  called  amebadiastase.  Ac- 
cording to  Metchnikoff,  the  digestion  of  erythrocytes  and  tissue  frag- 
ments is  accomplished  through  an  enzyme  of  the  macrophages  called 
macrocytase;  that  of  bacteria  or  other  substances  engulfed  by  micro- 
phages  by  a  similar  enzyme  called  microcytase. 

Following  the  general  law  that  living  protoplasm  cannot  be  digested, 
we  are  confronted  with  the  very  important  question  as  to  whether  living 
bacteria  are  engulfed  by  phagocytes  or  whether  they  are  first  destroyed 
by  extracellular  agencies  before  they  undergo  phagocytosis. 

Endolysins. — It  seems  positively  established  at  the  present  time 
that  leukocytes  do  take  up  living  bacteria,  which  may  either  grow  in- 
side the  leukocyte  or  be  destroyed  by  intracellular  substances  called 
endolysins.  On  the  other  hand,  leukocytes  do  not  take  up  extremely 
virulent  bacteria,  hence  the  question  arises  as  to  the  importance  of 
substances  in  the  body-fluids  which  neutralize  the  repelling  substances 
of  bacteria  and  facilitate  phagocytosis.  This  subject,  which  has  con- 
siderably modified  Metchnikoff's  views  of  phagocytosis,  will  be  con- 
sidered in  a  succeeding  chapter.  It  will  suffice  here  to  state  that  leuko- 
cytes may  engulf  living  bacteria  possessing  some  virulence,  for  not 
infrequently  an  infection  may  be  spread  by  bacteria  transported  into 
deeper  tissues  by  phagocytes,  when  they  resist  the  germicidal  activity 
of  the  endolysins,  bring  about  the  death  of  the  phagocyte,  and  are  thus 
liberated  into  new  tissues. 

Death  of  the  engulfed  bacteria  is,  therefore,  brought  about  by  en- 
dolysins 2  that  are  probably  different  from  the  digestive  enzymes.  These 
substances  are  strongly  bactericidal,  and  have  a  complex  structure  re- 
sembling bacteriolysins.  According  to  Weil,3  they  are  not  specific. 
They  are  resistant  to  65°  C.  or  even  higher,  do  not  readily  pass  through 
porcelain  filters,  are  precipitated  by  saturation  with  ammonium  sul- 
phate, and  resemble  the  enzymes  in  many  respects  (Manwaring4).  It 
is  probable  that  the  endolysins  act  not  only  upon  bacteria  that  have 
been  phagocytosed,  but  also  upon  free  bacteria  when  liberated  through 
disintegration  of  the  leukocytes.  In  this  manner  the  endolysins  would 
closely  resemble  the  normal  opsonins  or  bacteriolysins,  and  support  the 
contention  of  Metchnikoff  that  these  important  substances  contained 

1  Compt.  rend,  de  FAcad.  Sci.  de  Paris,  1901,  cxxxiii,  244. 

2  For  general  review,  see  Kling:  Zeit.  Immunitate,  1910,  7,  1. 

3  Arch.  f.  Hyg.,  1911,  74,  289.  «  Jour.  Exper.  Med.,  1912,  16,  250 


186  PHAGOCYTOSIS 

in  the  body  fluids  are  derived  primarily  from  the  cells  which  he  has 
classed  as  phagocytes.  According  to  Schneider,1  lymphocytes  and 
macrophages  seem  to  contain  little  or  no  endolysin,  and  these  cells  are 
not  so  active  in  the  phagocytosis  of  virulent  bacteria  as  are  the  micro- 
phages. 

It  is  possible,  however,  that  in  certain  instances  cells  not  only  fail 
to  kill  the  microparasites  they  ingest,  but  actually  protect  them  from 
circulating  antibodies;  apparently  the  microorganisms  of  leprosy, 
tuberculosis,  gonorrhea,  and  leishmaniosis  may  live  more  or  less  habit- 
ually within  tissue  cells,  and,  as  demonstrated  by  Roux  and  Jones,2 
living  phagocytes  are  able  to  protect  ingested  bacteria  from  the  destruc- 
tive substances  in  the  surrounding  fluid,  and  even  from  a  potent  homol- 
ogous antiserum. 

Indigestible  substances,  if  chemically  inert,  may  remain  in  cells, 
particularly  in  fixed  tissue-cells,  for  variable  periods  of  time.  The  leu- 
kocytes seem  to  transfer  such  particles  to  other  tissues,  particularly  to 
the  lymph-glands.  It  is  probable  that  these  phagocytes  are  in  turn 
engulfed  by  the  endothelial  cells.  Macrophages  of  the  lymph-sinuses  or 
the  leukocytes  may  be  destroyed  in  the  glands,  and  their  contents  re- 
phagocyted  by  these  cells.  In  just  what  manner  these  insoluble  par- 
ticles reach  the  gland  stroma  or  perilymphangeal  tissues  is  unknown; 
it  is  probable  that  they  are  liberated  from  the  lining  endothelial  cells, 
and  are  again  seized  by  the  young  connective-tissue  cells. 


THE  RELATION  OF  THE  BODY-FLUIDS  TO  PHAGOCYTOSIS 
Important  and  far-reaching  as  were  Metchnikoff's  researches  and 
conclusions,  they  were  not  allowed  to  pass  unchallenged,  especially  by 
the  adherents  of  the  humoral  school,  who  were  able  to  show  the  potent 
influences  of  the  body-fluids  in  the  mechanism  of  recovery  from  in- 
fections where  phagocytosis  was  little  in  evidence,  or,  indeed,  where 
phagocytosis  was  impossible.  It  was  shown  that  Metchnikoff's  original 
theory  was  untenable,  and  that  the  leukocyte  is  almost  impotent  if  re- 
moved from  the  influence  of  the  body-fluids. 

As  demonstrated  by  Denys,  Leclef,  Fliigge,  Nuttall,  Pfeiffer,  and 
others,  bacteria  may  be  killed,  i.  e.,  may  undergo  a  process  of  lysis  or 
disintegration,  by  means  of  substances  in  the  blood-serum  entirely  in- 
dependent of  phagocytosis  Later  researches  by  Wright,  Neufeld,  and 
their  coworkers  demonstrated  most  clearly  that  even  in  those  infections 

1  Arch.  f.  Hyg.,  1909,  70,  40.  2  Jour.  Exper.  Med.,  1916,  23,  601. 


RELATION    OF   THE    BODY-FLUIDS   TO    PHAGOCYTOSIS  187 

in  which  phagocytosis  was  observed  and  known  to  be  of  great  importance, 
the  bacteria  are  first  prepared  for  phagocytosis  by  substances  in  the 
body-fluids,  and  that  without  this  preliminary  preparation  of  the  bac- 
teria phagocytosis  was  slight  and  of  little  consequence. 

Metchnikoff  corroborated  most  of  these  discoveries,  and  modified 
his  theory  from  time  to  time  to  meet  the  developments  and  keep  them 
within  the  limits  of  the  phagocytic  theory.  For  example,  when  bac- 
teriolysis was  shown  by  Bordet  to  be  due  to  two  separate  substances  in 
the  body-fluids,  which  he  called  substance  sensibilisatrice  and  alexin 
(later  renamed  by  Ehrlich  amboceptor  and  complement  respectively), 
Metchnikoff  claimed  that  this  phenomenon  was  extracellular  digestion, 
similar  to  the  intracellular  digestion  that  occurs  within  the  phagocyte, 
and  brought  about  by  ferments  secreted  and  liberated  from  leukocytes 
or  other  cells  classed  as  phagocytes.  He  regards  alexin  as  a  cytase  se- 
creted by  leukocytes,  or  liberated  upon  their  disintegration;  similarly 
the  substance  sensibilisatrice  is  regarded  as  a  free  ferment  (fixateur), 
derived  principally  from  leukocytes,  and  concerned  in  preparing  the 
bacterial  or  other  cell  for  the  digestive-like  action  of  the  cytase. 

Aside  from  these  free  ferments  that  are  capable  of  producing  extra- 
cellular lysis,  Metchnikoff  has  long  known  that  other  substances  that 
aid  phagocytosis  itself  may  be  present  in  the  body-fluids;  he  regards 
these  as  of  the  nature  of  stimulins,  or  substances  that  stimulate  leuko- 
cytes to  become  more  actively  phagocytic.  On  the  other  hand,  Leish- 
man,  Wright  and  Douglas,  Neufeld,  and  Rimpau,  Hektoen,  and  others 
have  clearly  demonstrated  that  they  facilitate  phagocytosis,  not  by 
stimulating  the  leukocytes,  but  rather  by  lowering  the  resistance  of 
bacteria  or  in  some  way  rendering  them  more  vulnerable  to  phagocytosis 
(opsonins,  bacteriotropins) . 

Thus  the  gap  between  the  original  cellular  theory,  which  ascribed 
protection  and  cure  to  phagocytosis  pure  and  simple,  and  the  humoral 
theory  (finally  summed  up  by  Ehrlich  in  his  side-chain  theory),  which 
ascribed  the  chief  and  primary  roles  to  substances  in  the  body-fluids, 
and  relegated  phagocytes  to  a  position  of  secondary  importance,  re- 
garding them  only  as  scavengers  that  remove  dead  or  disabled  micro- 
organisms, has  been  filled  with  discoveries  correlating  both  processes. 

The  vitality  of  the  leukocyte  is  to  be  regarded  as  important  in  the 
consideration  of  phagocytosis  as  a  means  of  defense.  While  the  body- 
fluids  are  acting  upon  the  invaders,  the  leukocytes  themselves  are  prob- 
ably undergoing  quantitative  and  qualitative  changes.  They  are  in- 
creasing in  numbers,  and,  as  Rosenow1  has  shown,  are  undergoing  more 
1  Jour.  Infect.  Dis.,  1906,  3,  683. 


188  PHAGOCYTOSIS 

specific  changes.  Thus,  for  instance,  the  leukocytes  from  a  pneumonia 
patient  were  found  more  vigorous  against  invasion  of  the  pneumococcus 
than  are  those  from  a  normal  person,  regardless  of  the  influence  of  serum. 
When  a  microparasite  is  ingested,  the  process  has  only  begun.  Un- 
less suitable  endolysins  are  present  and  the  endotoxin  is  absorbed  or 
otherwise  dealt  with,  and  unless  suitable  digestive  enzymes  are  secreted 
and  the  bacterium  is  dissolved,  the  process  is  useless,  or,  indeed,  if  viable 
bacteria  are  transported  to  other  parts  of  the  body,  it  may  be  dangerous. 


THE  REVISED  THEORY  AND  ROLE  OF  PHAGOCYTOSIS  IN  IMMUNITY 

As  previously  stated,  Metchnikoff  has  revised  his  theory  from  time 
to  time,  as  these  discoveries  were  made  on  the  influence  of  substances  in 
the  body-fluids,  not  only  upon  phagocytosis  itself,  but  also  upon  the 
processes  of  immunity  in  general.  He  would  regard  extracellular  cy- 
tolysis  (bacteriolysis,  hemolysis,  etc.)  as  due  to  the  same  ferments  that 
bring  about  the  destruction  and  solution  of  the  ingested  bacterium  or 
other  cell  within  the  phagocyte,  and,  further,  these  extracellular  fer- 
ments are  derived  from  the  cells  that  are  classed  as  phagocytes.  By  this 
method  of  reasoning  he  would  preserve  the  importance  of  the  phago- 
cytic  theory. 

In  local  infections  phagocytosis  is  usually  well  marked,  and  no  doubt 
plays  an  important  part  in  resistance  to  and  recovery  from  these  con- 
ditions. Recent  investigations  by  Bull1  have  shown  that  following 
agglutination  of  various  bacteria  in  vivo,  phagocytosis  of  the  micro- 
organisms frequently  follows.  In  infections  due  to  the  various  patho- 
genic micrococci,  as  staphylococci,  pneumococci,  and  streptococci, 
phagocytosis  appears  to  be  most  active  and  an  important  means  of 
overcoming  the  infection.  In  other  infections,  as  those  due  to  the  ty- 
phoid bacillus  and  allied  bacilli,  it  is  probable  that  extracellular  sub- 
stances or  antibodies  are  chiefly  operative  in  affording  protection  or  in 
overcoming  infection,  although  certain  of  these,  as  the  agglutinins  and 
opsonins,  facilitate  phagocytosis. 

The  question,  then,  of  the  relative  importance  of  the  cellular  and 
humoral  theories  of  immunity  resolves  itself  to  a  consideration  of  the 
origin  of  the  substances  in  the  body-fluids  so  potent  in  both  processes. 
If  they  are  derived  solely  from  the  cells  known  to  act  as  phagocytes,  then 
the  cellular  theory  of  phagocytosis,  in  its  broader  meaning,  is  the  one 
explanation  of  the  processes  of  immunity  as  they  are  now  understood. 
1  Jour.  Infect.  Dis.,  1915,  16,  109. 


THE    MECHANISM    OF   PHAGOCYTOSIS  189 

This,  however,  has  never  been  proved,  and  it  is  entirely  likely  that  these 
substances  are  products  of  a  general,  rather  than  of  a  more  restricted, 
cellular  activity,  so  that  ultimately  all  immunologic  processes  are  cellular 
in  origin.  For  this  reason  we  prefer  to  speak  of  the  phagocytic  cell  in  its 
relation  to  immunity  when  dealing  with  the  relation  and  activity  of  mi- 
crophages  and  macrophages  in  a  limited  sense  in  the  process  of  phago- 
cytosis. 

THE  MECHANISM  OF  PHAGOCYTOSIS 

Aside  from  the  question  of  whether  fixed  and  wandering 
cells  may  engulf  virulent,  microparasites  and  the  influence  of  sub- 
stances in  the  body  fluids  upon  this  phase  of  phagocytosis,  we  have 
for  consideration  the  mechanism  whereby  these  cells  engulf  bacteria 
and  other  microparasites,  various  cells,  and  inorganic  particles. 
Based  upon  the  direct  observations  of  Metchnikoff  and  his  pupils, 
the  phase  of  engulfing  is  accomplished  by  means  of  pseudopods 
and  the  ameboid  movement  of  the  phagocyting  cell,  whereby  the 
particles  become  adherent  and  are  finally  rolled  or  passed  into  the 
protoplasm  of  the  phagocyte.  Recent  investigations  by  Kite  and 
Wherry1  would  indicate,  however,  that  in  so  far  as  leukocytes  are  con- 
cerned these  processes  are  of  minor  importance,  and  that  phagocytosis  X 
depends  upon  the  physical  conditions  of  the  surface  of  the  phagocytic ' 
cells,  a  "stickiness"  of  the  leukocytes,  whereby  bacteria  and  other  par- 
ticles become  adherent,  the  engulfing  being  a  purely  passive  process 
which  depends  upon  protoplasmic  streaming  within  the  cells.  They 
believe  that  such  substances  as  opsonins  act  in  increasing  phagocytosis 
merely  because  they  increase  the  ".stickiness"  of  the  cells,  and  that 
phagocytosis  depends  essentially  upon  the  relative  stickiness  of  phago- 
cytes and  bacteria;  increased  phagocytosis  in  the  presence  of  serum  and 
particularity  unheated  serum  is  ascribed  to  an  increased  stickiness  of 
the  leukocytes.  Mechanical  contact  as  well  as  certain  variations  in 
chemical  reactions  may,  therefore,  result  in  the  production  of  those 
changes  in  contour  and  protoplasmic  streaming  responsible  for  rolling 
the  particle  within  the  protoplasm  of  the  cell. 

1  Jour.  Infect.  Dis.,  1915,  16,  109. 


CHAPTER  XI 

OPSONINS 

Historic. — Although  there  can  be  no  doubt  as  to  the  importance  of 
phagocytosis  in  the  mechanism  of  recovery  from  infection,  yet  it  was 
shown  by  Metchnikoff,  as  early  as  1893,  that  the  body-fluids  contained 
substances  that  greatly  facilitated  the  phagocytic  process,  and  that 
leukocytes  removed  from  this  influence  were  practically  powerless  to  en- 
gulf and  destroy  the  invading  bacterium.  In  other  words,  if  leukocytes 
and  bacteria  are  washed  free  from  all  traces  of  serum  and  then  mixed, 
very  few  of  the  leukocytes  will  be  found  capable  of  phagocytizing  the 
bacteria,  which  means  that  spontaneous  phagocytosis  is  feeble  and  hence 
of  slight  importance.  When,  however,  fresh  serum  is  added,  especially 
the  serum  of  an  animal  immunized  against  the  microorganism  used  in 
the  experiment,  phagocytosis  is  marked,  and,  indeed,  most  impressive. 
Metchnikoff  attributed  this  difference  to  the  influence  of  a  substance 
in  the  serum  that  stimulated  (stimulins)  the  leukocytes  to  become  phag- 
ocytes, but  later  researches  have  shown  that  this  is  probably  erroneous,  and 
that  the  serum  facilitates  phagocytosis  not  by  exerting  a  stimulating  influ- 
ence upon  the  leukocytes,  but  by  preparing  the  bacteria  for  the  process  by 
making  them,  as  it  were,  more  attractive  to  the  leukocytes. 

Denys  and  Leclef,  in  1895,  were  among  the  first  to  demonstrate 
the  effect  of  serum  on  bacteria  in  the  process  of  phagocytosis,  and  the 
fact  that  the  active  substance  was  not  bactericidal  in  action,  but  in  the 
nature  of  a  new  antibody.  Since  Metchnikoff  had  shown  that  freshly 
isolated  or  virulent  strains  of  bacteria  were  not  readily  phagocytized, 
but  seemed  to  resist  or  repel  the  leukocytes,  it  was  natural  for  these 
observers  to  suggest  that  the  action  of  this  substance  in  serum  was  to 
neutralize  the  exotoxins  and  endotoxins  of  microorganisms  that  were 
regarded  as  responsible  for  negative  chemotactic  influences,  and  thus, 
by  robbing  them  of  at  least  two  defensive  weapons,  prepare  them  for 
phagocytosis. 

The  subject  remained  in  an  uncertain  state  until  1903,  when  Wright, 
and  later  Wright  and  Douglas,  demonstrated  more  clearly  this  action  of 
serum  upon  bacteria  in  aiding  phagocytosis.  Using  their  own  modifica- 
tion of  the  technic  devised  by  Leishman  for  measuring  the  phagocytic 

190 


PROPERTIES   AND   NATURE    OF   OPSONINS  191 

power  of  the  blood,  these  observers  first  determined  the  direct  depend- 
ence of  phagocytosis  upon  some  ingredient  of  the  blood-serum,  and  fur- 
ther proved  that  this  substance  acts  directly  upon  bacteria,  is  bound  by 
the  bacteria,  and  renders  them  more  easily  ingested  by  the  leukocytes, 
i.  e.,  more  readily  phagocytable.  To  this  substance  they  gave  the  name 
opsonin  (from  opsono,  I  prepare  food  for).  At  the  same  time,  and  in- 
dependently of  Wright,  Neufeld  and  Rimpau  conducted  similar  inves- 
tigations with  immune  serum  and  reached  similar  conclusions,  but  called 
the  substance  bacteriotropin.  Since  then  both  terms  have  been  used, — 
the  former  more  frequently  in  English  literature, — and  this  is  permissible, 
providing  that  it  is  understood  that  both  are  practically  the  same  anti- 
body, and  not  distinct  and  separate  from  each  other. 

As  will  readily  be  understood,  the  bacterial  opsonins  have  been 
studied  most  extensively,  but  opsonins  may  be  present  in  normal  and 
immune  serums  for  other  cells,  such  as  erythrocytes,  and  these  hemopso- 
nins  are  regarded  as  separate  antibodies,  distinct  from  hemagglutinins 
and  hemolysins. 

Definition. — Opsonins  are  substances  in  normal  and  immune  serums 
which  act  upon  bacteria  and  other  cells  in  such  a  manner  as  to  prepare  them 
for  more  ready  ingestion  by  the  phagocytes. 

Properties  and  Nature  of  Opsonins. — There  is  considerable  differ- 
ence of  opinion  regarding  the  identity  and  probable  structure  of  opsonins 
in  normal  and  immune  serums.  Just  as  agglutinin  for  a  bacterium, 
such  as  Bacillus  typhosus,  may  be  found  in  varying  amounts  in  normal 
serum,  so  various  opsonins  for  different  bacteria  may  be  found  in  normal 
serums.  These  normal  opsonins  appear  more  or  less  specific  for  a  given 
bacterium,  and  in  immune  serum  the  specific  opsonic  substance  for  the 
particular  bacterium  or  cell  with  which  immunization  has  been  pro- 
duced is  developed  to  a  high  degree.  Both  owe  their  full  effect  to  the 
interaction  of  two  substances.  One  of  these,  the  common  substance, 
is  thermolabile,  and  destroyed  by  heating  the  serum  to  from  56°  to  58°  C. 
for  half  an  hour,  whereas  the  other  more  specific  substance  remains  un- 
affected. The  latter,  in  both  normal  and  immune  serums,  is  opsonic 
by  itself,  although  in  the  absence  of  the  common  thermolabile  substance, 
to  a  less  degree,  and  is  produced  anew  and  specifically  by  artificial  im- 
munization or  as  the  outcome  of  spontaneous  infections. 

The  true  nature  of  opsonins  is,  therefore,  difficult  to  understand. 
They  have  been  compared  to  receptors  of  the  second  order,  with  a  hap- 
tophore  and  a  toxophore  or  opsoniferous  group.  Receptors  of  this 
order,  however,  are  active  and  independent  of  the  presence  or  absence 


192  OPSONINS 

of  complement,  whereas  the  opsonins,  although  active  to  some  extent 
in  the  absence  of  complement,  are  far  more  so  if  a  complement  is  present. 
In  this  respect  they  resemble  amboceptors,  or  receptors  of  the  third 
order,  opsonins  in  normal  and  immune  serums  representing  respectively 
normal  and  immune  bacterial  amboceptors.  One  objection  to  this 
view  of  their  structure  is  their  activity,  however  slight,  when  the  ther- 
molabile  substance  is  removed  by  heating,  unless  the  amboceptors  are 
complemented  by  an  endocomplement,  as  from  the  bacteria  themselves. 

At  the  present  time,  therefore,  not  a  few  observers  doubt  that  opso- 
nins exist  as  true  and  separate  antibodies,  and  are  inclined  to  regard 
thermolabile  opsonin  (largely  the  opsonin  in  fresh  normal  serum)  as  a 
complement,  and  thermostabile  opsonin  (largely  immune  opsonin  or 
bacteriotropin)  as  an  amboceptor;  it  would  appear  that  either  alone, 
but  more  especially  the  latter,  may  facilitate  phagocytosis  to  some  ex- 
tent. This  process  is,  however,  much  more  marked  when  both  sub- 
stances are  acting  in  unison.  While  it  is  true  that  the  bacteriolysin 
and  opsonin  content  of  a  serum  do  not  run  parallel,  our  methods  for 
measuring  these  are  not  entirely  satisfactory;  both  intracellular  and 
extracellular  lysis  may  be  mere  differences  in  degree,  depending  upon 
the  nature  of  the  bacterium  or  the  concentration  of  the  antibodies, 
rather  than  upon  separate  and  distinct  antibodies. 

Source  of  Opsonins. — Little  is  definitely  known  regarding  the  source 
of  opsonin.  Thermostabile  opsonin — that  which  is  increased  by  arti- 
ficial immunization  or  during  disease,  and  is  largely  in  the  nature  of  an 
amboceptor — is  probably  a  product  of  general  cellular  activity,  and 
especially  of  the  local  cells  at  the  site  of  infection.  Thermolabile  opsonin 
— largely  the  opsonin  occurring  in  normal  serum,  and  in  the  nature  of  a 
complement — is  probably  a  product  of  the  leukocytes  and  other  cells  as 
well,  as  it  has  never  been  proved  that  the  leukocytes  are  the  sole  source 
of  the  complements,  as  Metchnikoff  would  have  us  believe. 

Susceptibility  to  Opsonification. — As  previously  stated  in  the  chap- 
ter on  infection,  not  all  bacteria  are  equally  susceptible  to  opsonifica- 
tion.  As  a  general  rule,  recently  isolated  and  virulent  microorganisms 
resist  the  influence  of  opsonins  until  they  have  undergone  culture  sev- 
eral times.  This  resistance  may  be  due  to  capsule  formation,  thicken- 
ing of  the  ectoplasm,  actual  self-immunization  of  the  bacterium,  or  the 
influence  of  endotoxins  as  a  protective  means  against  the  antibodies  of  a 
host,  all  of  these  being  weakened  or  lost  upon  artificial  culture-media. 

Effect  of  Opsonins  on  Bacteria. — We  know  nothing  definite  regard- 
ing the  manner  in  which  opsonins  prepare  bacteria  for  phagocytosis 


ROLE   OF   OPSONINS   IN   IMMUNITY  193 

except  that  opsonification  in  itself  apparently  does  not  impair  the  vital- 
ity of  the  bacterium,  in  so  far,  at  least,  its  viability  is  concerned. 

Role  of  Opsonins  in  Immunity. — Although  the  exact  identity  of 
normal  and  immune  opsonins  and  their  relation  to  other  antibodies  is  as 
yet  unsettled,  the  important  relation  they  bear  to  processes  of  immunity 
is  generally  recognized,  especially  their  ability  in  aiding  resistance  to 
infection  by  facilitating  phagocytosis.  They  are  operative  in  some  in- 
fections more  than  in  others,  and  they  are  especially  active  in  those  con- 
ditions in  which  phagocytosis  is  recognized  as  the  chief  defensive  force, 
as,  for  example,  in  pyogenic  infections.  In  these  conditions  their  pres- 
ence has  been  taken  as  a  measure  (opsonic  index  of  the  resistance  of  the 
host)  and,  largely  through  the  researches  of  Wright  and  Douglas,  a 
technic  for  detecting  their  presence,  kind,  and  quantity  in  the  body- 
fluids  has  been  devised,  the  method  and  information  it  yields  being  of 
value  under  certain  limitations  and  in  some  infections.  (See  next 
chapter.) 

If  experiments  in  vitro  may  be  taken  as  an  example  of  what  occurs 
in  vivo,  it  must  be  true  that  leukocytes  are  capable  of  consuming  an 
enormous  number  of  bacteria.  Experiments  with  washed  leukocytes 
—those  removed  from  the  influence  of  serum — show  that  spontaneous 
phagocytosis  is  very  slight.  Metchnikoff  declared  these  experiments  to 
be  untrustworthy  for  the  reason  that  the  various  manipulations  of  wash- 
ing injures  the  vitality  of  the  leukocytes.  When,  however,  bacteria  are 
opsonized,  that  is,  are  exposed  to  a  serum  containing  opsonins,  and  then 
are  thoroughly  washed,  it  is  found  that  the  washed  leukocytes  engulf 
enormous  numbers  of  bacteria,  showing  that  Metchnikoff's  objection  to 
these  experiments  is  unwarranted.  Granting,  then,  that  what  we  call 
opsonins  are  substances  that  facilitate  phagocytosis,  and  that  phago- 
cytosis is  a  process  of  great  importance,  especially  in  certain  infections, 
we  must  conclude  that  opsonins  play  a  very  important  role  in  immunity; 
in  fact,  they  constitute  the  very  basis  of  the  phenomenon  of  phagocytosis 
in  the  broader  meaning  of  the  term. 
13 


CHAPTER  XII 
OPSONIC  INDEX 

WHETHER  opsonins  are  regarded  as  separate  antibodies  or  as  being 
identical  with  complements  and  amboceptors,  a  measure  of  their  quan- 
tity and  power  may  be  of  aid  in  formulating  a  diagnosis,  as  a  guide  to 
active  immunization,  and  as  one  means  of  determining  the  potency  of 
various  immune  serums  used  for  therapeutic  purposes,  such  as  antimen- 
ingococcus  and  antipneumococcus  serums.  We  are  mainly  indebted 
to  Leishman,  Wright  and  Douglas,  Neufeld  and  Rimpau,  and  their 
coworkers  for  devising  a  technic  that,  however  imperfect  it  may  be  ac- 
cording to  the  results  obtained,  has  opened  a  new  and  important  field 
for  the  study  of  immunologic  processes. 

Principle. — This  is  based  upon  the  method  devised  by  Wright  and 
Douglas,  whereby  it  was  sought  to  determine  the  amount  and  kind  of 
opsonin  in  a  patient's  serum  by  comparing  the  degree  of  phagocytosis 
with  that  occurring  when  normal  serum  was  used. 

Definition. — The  opsonic  index  is  the  ratio  of  the  number  of  bacteria 
ingested  by  a  given  number  of  phagocytes  in  the  presence  of  a  patient's  serum, 
to  the  number  ingested  by  the  same  number  of  phagocytes  in  the  presence  of 
normal  serum. 

"An  equal  volume  of  the  patient's  serum,  measured  in  a  capillary 
pipet,  is  mixed  with  an  equal  volume  of  a  suspension  of  washed  leuko- 
cytes derived  from  a  normal  blood.  After  this  'phagocytic  mixture' 
has  been  digested  for  a  suitable  period  at  37°  C.,  film  preparations  are 
made  and  stained. 

"A  'phagocytic  count'  is  then  undertaken,  i.  e.,  the  average  bacterial 
ingest  of  the  leukocytes  in  the  phagocytic  mixture  is  determined,  and 
this  is  compared  with  the  average  ingest  of  the  leukocytes  in  a  phago- 
cytic mixture  made  with  normal  blood. 

"The  expression  thus  obtained, 

Average  ingest  of  the  individual  phagocyte  in  the  mixture  containing  the 

patient's  serum. 

Average  ingest  of  the  individual  phagocyte  in  the  mixture  containing  nor- 
mal serum  is  denoted  the  opsonic  index"  (Wright). 

194 


LIMITATION    OF   THE    METHOD  195 

Purpose  of  the  Method. — The  opsonic  index  aims  to  serve  as  a  guide: 

1.  In  diagnosing  the  presence  of  bacterial  infection,  or  rather  in  dis- 
covering whether  the  natural  protective  powers  of  the  patient's  blood 
have  been  diminished  or  increased  as  the  result  of  the  immunizing  in- 
fluence of  the  infection. 

2.  In  connection  with  vaccine  therapy,  to  guard  against  diminishing 
the  opsonin  content  of  the  patient's  blood;  to  assure  ourselves  that 
our  efforts  to  increase  them  have  been  successful,  and  occasionally  to 
ascertain  how  long  the  store  of  opsonin  that  has  been  obtained  for  the 
patient  remains  in  the  blood. 

Limitation  of  the  Method. — In  ascertaining  the  opsonic  index  of  a 
patient's  serum,  we  must  take  it  for  granted — although  it  has  not  been 
proved : 

1.  That  the  bacteria  act  the  same  in  the  body  as  they  do  in  the  test- 
tube.     This  is  known  not  to  be  the  case,  for  virulent  organisms  resist 
phagocytosis,  whereas  a  non-virulent  strain  of  the  same  bacterium  is 
easily  phagocyted.     If,  therefore,  a  laboratory  culture  of  attenuated 
organisms  is  used  in  making  the  opsonic  index,  the  result  can  hardly 
be  accepted  as  a  criterion  of  the  power  of  the  patient  to  overcome  the 
"resistant"  or  more  virulent  organism  as  it  occurs  in  the  body.     This 
source  of  error  can  be  overcome  in  a  manner  if  the  microorganism  is 
isolated  and  used  at  once  before  attenuation  occurs. 

2.  That  the  leukocytes  are  a  constant  factor,  and  need  not  be  taken 
into  account.     Investigation  has  shown  that,  as  a  result  of  infection, 
the  leukocytes  probably  undergo  qualitative  changes  and  it  is  hardly 
fair  to  accept  phagocytosis  by  normal  leukocytes  as  a  criterion  of  pha- 
gocytosis with  the  patient's  own  leukocytes,  as  it  occurs  in  the  body  dur- 
ing the  infection. 

3.  The  method  assumes  that  phagocytosis  by  the  polynuclear  leuko- 
cyte plays  a  large  part  in  overcoming  the  infection.     In  many  cases, 
however,  this  is  by  no  means  proved.     For  example,  in  tuberculosis 
it  is  not  this  form  of  leukocyte,  but  the  mononuclear  form  or  the  lympho- 
cyte, which  seems  to  be  more  important,  and  hence  it  is  difficult  to  un- 
derstand how  the  index  with  the  polynuclear  leukocyte  can  aid  the 
question  of  diagnosis  or  treatment. 

4.  The  chances  for  error  are  considerable.     To  be  of  any  value,  the 
work  requires  experience  and  painstaking  care.     The  results  obtained 
by  competent  workers  with  the  same  blood  may  show  variation,  but 
it  must  be  said  that,  with  strict  attention  to  technic  and  insistence  upon 
perfect  preparations,  the  worker  may  usually  obtain  valuable  results. 


196  OPSONIC    INDEX 

An  index  taken  at  one  time  by  one  person  and  later  by  another,  and  so 
on,  will  not  be  of  as  much  value  as  when  all  are  taken  by  the  same  worker, 
who  brings  practice,  skill,  and  conscientious  care  to  his  aid. 

Precautions  in  Technic. — 1.  Proper  controls  should  be  used.  When 
dealing  with  the  tubercle  bacillus,  the  staphylococcus,  or  any  other 
saprophyte  of  the  external  surfaces,  or  with  any  pathogenic  organism 
with  which  we  have  normally  no  relations,  the  serum  of  a  normal  in- 
dividual or  the  mixed  serum  of  a  number  of  normal  persons  will  furnish 
the  standard  of  comparison.  When,  on  the  other  hand,  we  are  dealing 
with  intestinal  bacteria  or  with  a  saprophyte  of  the  mucous  membrane, 
where,  as  a  rule,  any  relation  with  them  will  be  denied,  it  is  difficult  to 
establish  a  standard  of  health.  Pooled  serum  is,  therefore,  necessary, 
and  will  furnish  a  standard  for  comparison  for  the  purpose  of  measuring 
the  fluctuations  that  may  occur  in  the  patient's  blood. 

2.  A  reasonable  degree  of  phagocytosis  should  occur  in  the  control 
serum.     This  is  one  of  the  main  drawbacks  to  the  value  of  the  method 
for  certain  pathogenic  organisms,  as  the  pneumococcus,  meningococcus, 
streptococcus,  etc.,  may  resist  phagocytosis  in  normal  serum,  and  thereby 
show  abnormally  high  indices  with  immune  serum. 

3.  Efforts   at   spontaneous   phagocytosis    should  be  suppressed  in 
order  to  measure  more  accurately  the  opsonin,  as  shown  by  the  degree 
of  phagocytosis  independent  of  the  inherent  activities  of  the  cell  itself. 
Spontaneous  phagocytosis  can  largely  be  overcome  by  using  1  per  cent, 
solution  of  sodium  citrate  such  as  is  used  for  the  collection  of  leukocytes. 

4.  The  ingest  of  a  sufficiently  large  number  of  phagocytes  should  be 
counted.     As  a  general  rule,  100  cells  should  represent  the  minimum. 


TECHNIC 
The  necessary  constituents  for  making  the  test  are  as  follows: 

1.  The  patient's  serum  and  normal  serums  to  serve  for  the  control. 

2.  A  bacterial  emulsion. 

3.  A  suspension  of  washed  human  leukocytes  in  normal  salt  solution. 
Collection  of  Patient's  and  Control  Serum. — 1.  The  blood  is  col- 
lected in  a  Wright  capsule,  as  described  in  Chapter  II. 

2.  Care  must  be  taken  not  to  heat  the  blood  when  sealing  the  tube. 
In  drawing  off  the  serum,  avoid  an  admixture  of  corpuscles,  as  these  may 
lower  the  opsonic  index. 

3.  If  coagulation  is  incomplete  or  the  serum  has  not  been  well  sep- 
arated, the  clot  may  be  broken  up  gently  with  a  platinum  wire  and  the 


TECHNIC 


197 


tubes  centrifuged.     Slight  discoloration  of  the  serum  from  mechanically 
breaking-up  a  few  erythrocytes  will  not  interfere  with  the  test. 

4.  If  gross  contamination  is  avoided,  blood  may  be  kept  for  one  or 
two  days  in  a  dark  place  without  much  deterioration  of  its  opsonin 
content. 

5.  It  is  always  well  to  collect  the  control  bloods  at  the  same  time  the 
patient's  blood  is  taken,  or,  if  this  cannot  be  done,  to  place  them  in  an 
ice-chest  as  soon  after  collection  as  possible.     When  conducting  the 
test,  the  control  serums  are  pooled  and  mixed  in  a  clean  watch-glass. 

The  Bacterial  Emulsion. — 1.  This  must  be  perfectly  uniform,  free 
from  clumps,  and  must  not  undergo  agglutination,  either  spontaneous 
or  with  the  serum  to  be  tested. 

With  many  bacteria,  especially  the  motile  ones,  such  as  Bacillus 
coli  and  Bacillus  typhosus,  it  is  comparatively  easy  to  secure  a  uniform 
emulsion.  Staphylococci,  streptococci,  and  pneumococci,  as  a  rule, 
present  no  difficulties.  After  growing  the  culture  for  from  eighteen  to 
twenty-four  hours  on  slants  of  a  suitable  medium,  add  three  cubic  centi- 
meters of  sterile  1  per  cent,  salt  solution,  and  gently  remove  the  bac- 
terial growth  with  a  platinum  loop.  The  mixture  is  then  pipeted  into  a 
separate  flask  or  thick  glass  test-tube  containing  glass  beads,  and  shaken 
by  machine  or  by  hand  until  it  is  thoroughly  emulsified.  If  necessary, 
the  emulsion  may  be  centrifugalized  to  remove  clumps  and  is  then  ready 
for  use. 

2.  It  must  consist  of  bacteria  that  stain  evenly  and  well. 

Only  young  cultures  should  be  used;  for  example,  an  eighteen  to 
twenty-four-hour  culture  of  freely  growing  organisms  and  a  seven  days' 
growth  of  tubercle  bacillus. 

3.  It  must  be  of  such  strength  as  to  give  a  leukocytic  ingest  that  will 
enable  adequate  differentiation  to  be  made  of  the  opsonin  content  of  the 
various  serums. 

An  emulsion  that  does  not  yield  a  count  of  at  least  250  bacteria 
within  100  leukocytes  is  too  weak  to  yield  a  satisfactory  differential 
count.  If  the  emulsion  is  too  thick,  bacteria  overlie  the  leukocytes 
and  introduce  error.  As  a  general  rule,  a  suspension  containing  500,- 
000,000  bacteria  per  cubic  centimeter  is  satisfactory.  Experience  will 
teach  the  right  density  to  be  used,  and  frequent  trials  may  be  necessary 
before  the  right  one  is  secured. 

4.  The  bacteria  must  be  such  as  will  not  prove  seriously  dangerous. 
In  order  to  obviate  any  danger  attending  work  with  such  organisms  as 
the  glanders  and  the  tubercle  bacillus,  first  kill  the  culture  by  pouring 


198  OPSONIC    INDEX 

on  a  10  to  40  per  cent,  solution  of  formalin,  mix  the  culture,  shake, 
transfer  to  a  centrifuge  tube,  and  centrifugalize  until  the  bacteria  have 
been  carried  to  the  bottom  of  the  tube.  Pipet  off  the  supernatant 
formalin,  wash  in  normal  salt  solution,  centrifuge,  pipet  off  again,  and 
finally  mix  the  sediment  in  sufficient  salt  solution  to  make  a  satisfactory 
suspension. 

The  Washed  Leukocytes. — These  should  consist  mainly  of  the  poly- 
nuclear  leukocytes  of  a  healthy  person  washed  free  from  any  admixture 
with  serum.  As  usually  obtained,  the  leukocytes  are  mixed  with  red 
corpuscles.  It  is  necessary  to  collect  blood  for  the  leukocytic  mixture 
from  a  person  whose  corpuscles  are  known  to  be  insensitive  to  agglutina- 
tion, as  otherwise  there  is  an  undue  lowering  of  the  opsonic  effect. 

1.  Place  4  c.c.  of  sterile  2  per  cent,  solution  of  sodium  citrate  in  dis- 
tilled water  in  sterile  10  c.c.  centrifuge  tubes. 

2.  Prick  the  finger,  and  add  1  c.c.  of  blood.     Agitate  well  to  insure 
thorough  mixing. 

3.  Centrifuge  at  a  sufficiently  high  speed  to  mix  the  red  corpuscles 
and  leukocytes  at  the  bottom  of  the  tube  and  avoid  clumping  of  the 
leukocytes. 

4.  Draw  off  the  supernatant  fluid,  add  5  c.c.  of  1  per  cent,  salt  solu- 
tion, mix,  and  centrifuge. 

5.  Wash  once  more.     Draw  off  the  supernatant  fluid. 

6.  Add  sufficient  salt  solution  to  bring  the  total  volume  up  to  that  of 
the  blood  originally  taken — i.  e.}  1  c.c.     Mix  well. 

The  Test. — 1.  Prepare  capillary  pipets  of  approximately  the  same 
caliber.  These  are  made  by  taking  6-inch  lengths  of  soft  clean  glass 
tubing  having  an  external  diameter  of  -f$  inch,  heating  them  in  the 
middle  in  the  tip  of  the  blow-pipe  or  the  Bunsen  flame  until  about  ^ 
inch  length  of  tubing  is  quite  soft.  Remove  from  the  flame,  and  by  rap- 
idly separating  the  two  hands  draw  out  the  molten  glass  to  a  length  of 
from  18  to  20  inches.  After  cooling,  the  capillary  thread  is  cut  across 
with  a  small  file,  so  that  from  6  to  8  inches  is  left  attached  to  each  piece 
of  tubing.  The  ends  must  be  cut  square,  as  ragged  and  uneven  ends 
are  difficult  to  handle.  By  means  of  a  wax-pencil  make  a  fine  mark  at  a 
point  about  an  inch  from  the  free  end  of  each  capillary  thread.  .This 
indicates  the  unit  volume  (Fig.  48). 

2.  Adjust  a  well-fitting  rubber  teat,  and  draw  up  the  unit  volume  of 
blood-cells.  A  tiny  bubble  of  air  is  now  allowed  to  enter  the  thread,  and 
then  one  volume  of  the  bacterial  emulsion  is  added;  another  air-bubble 
is  allowed  to  enter,  and  finally  one  volume  of  serum,  so  that  we  have 


TECHNIC 


199 


named  in  their  order  in  the  capillary  tube  from  above 
downward,  one  volume  of  blood-cells,  an  air-bubble, 
one  volume  of  bacterial  emulsion,  an  air-bubble,  and 
one  volume  of  serum  (Fig.  48) . 

3.  By  making  gentle  pressure  on  the  teat  these 
are  then  blown  out  on  the  surface  of  a  clean  glass 
slide,  and  perfect  mixture  effected  by  making  alter- 
nate aspiration  and  expulsion  from  the  capillary  tube 
at  least  six  times  (Fig.  49). 

4.  Carefully  reaspirate  into  the  capillary  thread, 
so  that  the  mixture  occupies  about  the  middle,. and 
seal  the  tip  in  a  low  Bunsen  flame  (Fig.  50) . 

5.  Remove  the  teat,  and  with  the  wax-pencil  mark 
the  tube  with  the  name  or  number  of  the  serum. 

6.  A  similar  preparation  is  prepared  with  the 
pooled  serum  (control). 

7.  The  phagocytic  mixtures  are  then  placed  in 
an  incubator  at  37°  C.  for  fifteen  minutes,  except  in 
the  case  of  such  bacteria  as  the  Bacillus  typhosus 
and  the  Bacillus  coli,  as  lysin  and  agglutinin  may 
be  present  in  the  serums  of  such  bacteria  when  the 
period   is   reduced   to    ten  minutes.     The    special 
opsoriic  incubators  built  to  accommodate  individual 
pipets  are  particularly  serviceable. 

8.  The  tubes  are  then  removed  from  the  incuba- 
tor, the  teats   readjusted,  the  tip  of   the  capillary 
threads  scratched  with  a  file,  and  evenly  broken  off. 
The  phagocytic  mixture  is  carefully  expelled  on  a 
clean  glass  slide,  and  a  perfect  mixture  made  by 
alternate    aspiration    and    expulsion.      Avoid    air- 
bubbles.      The   whole   is   then   reaspirated,  and   a 
small  drop  of  the  mixture  placed  on  each  of  two  clean 
slides  that  have  been  roughened  with  emery  paper 
about  %  inch  from  one  extremity.     With  the  edge 
of  a  spreader  slide  held  at  an  angle  of  about  30 
degrees,  and  with  moderate  pressure,   the  drop  is 
distributed   evenly  along   about   1J^  inches  of  the 
surface  of  the  slide  (Fig.  51).     The  smears  are  made 
in    duplicate,   because    one    may    be    more    nearly 
perfect  (Fig.  52)   than  the  other,   or  one  may  be 


i 


FIG. 

LARY       PlPET       FOR 

OPSONIC  INDEX  DE- 
TERMINATION. 


200 


OPSONIC    INDEX 


spoiled  in  the  staining,  when  the  second  may  be  utilized.  Each  slide 
is  then  labeled  at  one  end. 

9.  After  drying  in  the  air,  the  slides  must  be  fixed  and  stained. 

(a)  For  Ordinary  Bacteria. — 1.  Fix  by  covering  the  slide  with  a  sat- 
urated aqueous  solution  of  mercuric  chlorid  for  one  minute.  Wash  in 
water. 

2.  Cover  with  carbolthionin  and  stain  for  one  or  two  minutes, 
in  water. 


FIG.  49. — MIXING  THE  CONTENTS  OF  A  CAPILLARY  PIPET. 
Due  precautions  must  be  exercised  to  avoid  the  formation  of  bubbles. 

3.  Dry  thoroughly. 

(b)  For  Acid-fast  Bacilli. — 1.  Fix  by  inverting  the  films  for  thirty 
seconds  over  a  watch-crystal  or  jar  containing  formalin,  being  careful 
that  there  are  no  drops  of  formalin  on  the  edge  of  the  vessel  that  might 


TECHNIC 


201 


come  in  contact  with  the  preparation.  The  films  may  be  fixed  also 
with  a  saturated  solution  of  mercuric  chlorid  or  with  pure  methyl 
alcohol  for  two  minutes.  Wash  in  water  and  dry. 

2.  Heat  a  portion  of  carbolfuchsin  almost  to  boiling  in  a  test-tube, 
and  pour  the  hot  stain  over  the  films.  Allow  to  remain  for  at  least 
fifteen  minutes.  Wash  under  the  tap  and  dry. 


FIG.  50. — METHOD  OF  SEALING  A  CAPILLARY  PIPET. 

The  tip  of  the  pipet  is  placed  in  the  edge  of  a  flame.  The  teat  is  held  in  the 
same  position  uiitil  the  tip  has  been  sealed,  when  it  may  be  removed  without  disturb- 
ing the  contents  of  the  pipet. 

3.  Cover  with  a  5  per  cent,  solution  of  nitric  or  sulphuric  acid  for 
half  a  minute  or  longer  if  necessary,  until  decolorization  is  complete. 
Wash  thoroughly  under  the  tap. 


FIG.  51. — METHOD  OF  PREPARING  A  BLOOD  FILM. 

The  slide  is  laid  on  a  flat  surface;  the  drop  of  blood  is  placed  near  one  end;  the 
spreader  is  held  between  the  thumb  and  middle  finger  of  the  left  hand,  at  an  angle  of 
about  30  degrees,  and  quickly  pushed  to  the  opposite  end  of  the  slide. 

4.  Cover  with  4  per  cent,  aqueous  solution  of  acetic  acid  for  one  to 
two  minutes  to  remove  the  hemoglobin  from  the  red  cells.     Wash  and 
blot  lightly. 

5.  Cover  with  Loffler's  methylene-blue  for  two  minutes.     Wash  in 
water  and  dry  thoroughly  (Fig.  53). 


202  OPSONIC    INDEX 

10.  Examination  of  the  stained  films  with  the  oil-immersion  ob- 
jective of  the  microscope  will  show  that  polynuclear  leukocytes  have 
collected  more  toward  the  edges  and  the  end  at  which  the  spreading  was 
completed.     The  individual  leukocytes,  however,  should  be  separated 
from  one  another  (Figs.  54  and  55). 

11.  The  edge  of  the  film  is  examined,  and  the  number  of  bacteria 
found  in  each  series  of  five  consecutive  phagocytes  is  noted.     If  the 
technic  has  been  satisfactory,  no  great  divergence  should  be  found  in 
the  count  of  each  set  of  five  cells. 


FIG.  52. — BLOOD  FILMS  FOR  PHAGOCYTIC  COUNTS. 

The  first  slide  (on  the  extreme  left)  is  too  thick  and  honeycombed,  due  to  a 
greasy  slide  and  large  drop  of  blood;  the  second  is  likewise  thick  and  uneven;  the 
third  is  too  thin,  and  was  spread  with  too  small  an  amount  of  blood  and  with  the 
spreader  held  too  upright;  the  fourth  (extreme  right)  is  a  satisfactory  film;  it  was 
spread  on  a  clean  slide,  is  even,  smooth,  and  of  the  proper  thickness. 

12.  If  the  films  are  satisfactory,  divide  100  phagocytes  into  groups 
of  20.  The  average  ingest  of  each  group  should  not  show  a  difference  of 
over  10  per  cent.,  otherwise  the  technic  has  been  faulty  and  it  is  neces- 
sary to  count  250  phagocytes  or  to  repeat  the  test.  If  divergence  is 
due  to  the  fact  that  every  now  and  then  one  cell  has  a  considerable 
higher  ingest  than  others  and  the  bacteria  are  well  separated,  hyper- 
activity  of  the  cell  is  probably  the  cause.  If  the  bacteria  are  all  clumped 
together,  it  must  be  assumed  that  there  has  been  a  lack  of  care  in  prepar- 
ing the  bacterial  emulsion  or  that  agglutinin  is  present  in  the  serum, 
and  the  test  must  be  repeated  with  fresh  precautions. 


FIG.  53. — TUBERCLE  OPSONIC  INDEX. 

.  smear,  stained  after  the  method  given  in  the  text.     Case  IX,  C.  M.,  aged  twenty- 
two  years;   active  pulmonary  tuberculosis;  opsonic  index,  +1.6. 


FIG.  54. — AN  UNSATISFACTORY  FILM  FOR  A  PHAGOCYTIC  COUNT. 

Note  that  the  leukocytes  are  collected  in  masses  of  erythrocytes.     The  slide  was 

greasy  and  the  smear  too  thick. 


FIG.  55. — A  SATISFACTORY  FILM  FOR  A  PHAGOCYTIC  COUNT. 


QUANTITATIVE    ESTIMATION    OF    BACTERIOTROPINS  203 

13.  In  opsonic  work  the  question  as  to  how  a  certain  element  ought 
to   be   counted   becomes   quite   evident   and   important.     The  proper 
method  of  procedure  is  to  determine  definitely  how  they  may  best  be 
dealt  with,  and  then  to  follow  the  rule  adopted  consistently.     If  an 
organism  rests  on  the  border  of  a  cell,  it  will  be  better  to  consider  it  as 
within  the  cell  and  count  it.     Diplococci  or  division  forms  may  be 
counted  as  one  or  as  two,  provided  we  are  consistent  in  our  method.     In- 
dividual organisms,  as  distinguished  from  zoogleic  masses,  which  may 
be  lying  on  the  top  of  the  cell,  are  counted  as  if  they  were  within  the 
cell;  for  we  have  no  means  of  determining  definitely  whether  or  not  our 
suspicions  are  well  grounded.     In  the  case  of  a  beaded  or  vacuolated 
bacillus,  it  is  always  better  to  count  the  whole  element  as  a  unit. 

14.  The  phagocytic  index  is  the  average  number  of  bacteria  or  other 
cells  ingested  per  leukocyte  after  counting  at  least  from  50  to  100  cells. 
The  total  number  of  bacteria  ingested  is  divided  by  the  total  number  of 
phagocytes,  the  result  being  the  average  number  of  bacteria  ingested 
per  leukocyte — i.  e.,  the  phagocytic  index. 

15.  The  opsonic  index  is  then  given  by  the  ratio — 

Patient's  phagocytic  index 
Control    phagocytic    index 

For  example,  with  patient's  serum,  100  phagocytes  contain  300  bac- 
teria, the  phagocytic  index  being  -f-§-§-  =  3.  With  the  control  serum,  100 
phagocytes  contain  500  bacteria,  the  phagocytic  index  being  fw  =  5. 
The  opsonic  index  is : 

3  Patient's  phagocytic  index  _/\/» 
5  Control  phagocytic  index 

16.  Simon  and  Lamar  have  suggested  a  modification  of  Wright's 
method  that  has  been  adopted  by  many  laboratories.     It  consists  in 
diluting  the  patient's  and  control  serums  up  to  1 : 10  or  1 : 100,  and  pre- 
paring mixtures  of  various  dilutions  with  leukocytes  and  thinner  bac- 
terial emulsions.     The  percentage  of  phagocytic  cells  in  the  mixtures 
containing  the  serum  to  be  tested  is  compared  with  the  mixtures  con- 
taining normal  serum.     It  is,  therefore,  a  method  of  comparative  phago- 
cytic indices. 

QUANTITATIVE  ESTIMATION  OF  BACTERIOTROPINS  (NEUFELD) 
Of  the  various  methods  for  standardizing  an  immune  serum,  par- 
ticularly antimeningococcus  serum,  and  of  obtaining  some  idea  of  its 


204  OPSONIC    INDEX 

potency,  that  of  determining  the  bacteriotropic  or  opsonic  index  of  the 
serum  is  in  most  general  use. 

Neufeld's  technic  is  that  generally  employed,  and  is  similar  to  the 
serum  dilution  method  employed  by  Simon.  It  varies  from  the 
technic  of  Wright  in  several  particulars : 

1.  The  immune  serum  is  free  from  complement  (thermolabile  op- 
sonin). 

2.  The  actual  number  of  bacteria  within  the  leukocytes  are  not 
counted.     Various  dilutions  of  serum  are  used,  and  the  highest  dilution 
in  which  the  bacteria  are  ingested  in  great  numbers  is  compared  with  a 
normal  serum  in  similar  dilution  as  a  control.     The  highest  dilution  that 
still  favors  phagocytosis  is  then  taken  as  the  bacteriotropic  liter  of  the  serum. 

Serum. — The  serum  is  inactivated  by  heating  it  to  55°  C.  for  one- 
half  hour.  In  old  or  carbolized  serums  this  may  be  omitted,  as  they 
are  usually  free  from  complement.  Tuberculous  serums  also  should 
not  be  heated,  as  their  bacteriotropins  are  very  susceptible  to  heat. 

Normal  serum  from  an  animal  of  the  same  species  as  was  used  in  the 
preparation  of  the  immune  serum  should  be  used  as  the  normal  control. 

An  exactly  parallel  series  of  dilutions  with  normal  salt  solution  are 
made  of  the  immune  serum  and  pooled  normal  serums  in  a  series  of 
small  test-tubes.  At  least  0.5  c.c.  of  each  dilution  should  be  available 
for  the  test;  the  following  dilutions  may  be  used:  1:10,  1:20,  1:50, 
1:100,  1:200,  1:400,  1:600,  1:800,  1:1000,  1:2000,  etc.  After  working 
for  some  time  with  normal  serums  one  soon  learns  the  dilution  in  which 
the  normal  bacteriotropins  are  attenuated.  It  may  not  be  necessary, 
therefore,  to  use  the  whole  series  of  dilutions  with  the  normal  serum. 

Leukocytes. — Leukocytes  may  be  obtained  in  several  different  ways : 
(1)  By  injecting  a  guinea-pig  intraperitoneally  sixteen  to  twenty-four 
hours  previously  with  from  5  to  10  c.c.  of  sterile  aleuronat  solution  (for 
method  of  preparation  see  p.  361).  Pipet  the  peritoneal  exudate  into 
about  20  c.c.  of  sterile  1  per  cent,  sodium  citrate  in  normal  salt  solution 
in  centrifuge  tubes.  Centrifugalize,  and  wash  the  leukocytes  again 
three  times  with  sterile  normal  salt  solution.  The  sodium  citrate  solu- 
tion prevents  the  coagulation  and  formation  of  clumps  of  leukocytes. 

2.  Instead  of  aleuronat  a  sterile  25  per  cent,  solution  of  peptone 
may  be  injected  in  the  same  amount. 

'  3.  If  rabbit's  leukocytes  are  preferred,  10  c.c.  of  aleuronat  should  be 
injected  into  each  pleural  sac  or  20  c.c.  intraperitoneally.  For  mice, 
an  injection  of  1  c.c.  of  aleuronat  intraperitoneally  is  sufficient;  human 
leukocytes  may  be  obtained  after  the  method  of  Wright. 


QUANTITATIVE    ESTIMATION    OF    BACTERIOTROPINS  205 

4.  After  the  final  washing,  the  leukocytes  are  suspended  in  sufficient 
normal  salt  solution  until  an  opacity  equal  to  a  0.3  per  cent,  lecithin 
emulsion  in  salt  solution  is  attained. 

Culture. — Cultures  should  be  selected  with  great  care,  in  order  to 
avoid  using  one  that  displays  a  well-marked  tendency  to  undergo 
"spontaneous  phagocytosis,"  or,  on  the  other  hand,  one  unduly  re- 
sistant to  phagocytosis.  Usually  an  old  strain  of  meningococci  is 
serviceable;  it  is  generally  necessary  to  try  out  a  number  of  strains 
and  select  the  best. 

Meningococci  are  grown  for  twenty-four  hours  on  slants  of  glucose 
agar.  To  each  slant  add  0.5  c.c.  each  of  bouillon  and  of  normal  salt 
solution,  and  emulsify  the  growth.  Or  the  bacteria  may  be  employed 
in  the  form  of  a  sixteen  to  twenty-four  hour  homogeneous  broth  culture. 
Tubercle  bacilli  may  either  be  triturated  in  an  agate  mortar  with  1.5 
per  cent,  salt  solution  added  slowly  drop  by  drop,  or  the  tubercle  powder 
of  Koch  may  be  employed  in  an  emulsion  prepared  in  the  same  manner. 
The  resultant  emulsion  should  be  freed  from  coarser  clumps  by  brief 
centrifugalization,  but,  as  a  general  rule,  it  is  very  difficult  to  secure  a 
uniform  emulsion  of  tubercle  bacilli  by  any  method. 

The  Test. — 1.  The  mixtures  are  made  preferably  in  a  series  of  small 
test-tubes  about  5  cm.  long  and  1  cm.  wide. 

2.  Mix  0.1  c.c.  of  each  dilution  of  immune  serum  with  0.1  c.c.  of  bac- 
terial emulsion.     Plug  each  tube  with  cotton. 

3.  Mix  and  incubate  for  one  hour. 

4.  Add  0.1  c.c.  of  leukocytic  emulsion  to  each  tube.     Double  this 
quantity  may  be  used  if  the  emulsion  is  weak. 

5.  Mix  and  incubate  for  from  one-quarter  to  two  hours,  depending 
upon  the  variety  of  microorganism. 

6.  At  the  end  of  the  second  incubation  the  leukocytes  will  have 
settled  to  the  bottom  of  the  tubes.     Carefully  remove  the  supernatant 
fluid  from  each  tube;  mix  the  sediment  well  with  a  loop,  and  make  smears 
on  slides.    Label  each  slide  carefully  to  correspond  to  its  serum  dilution. 

7.  Dry  the  smears  in  the  air,  fix  with  methyl  alcohol,  and  stain  with 
carbolthionin,  as  previously  described. 

The  Controls. — 1.  A  series  of  the  lower  dilutions  of  pooled  normal 
serums  are  set  up  in  exactly  the  same  manner. 

2.  A  tube  containing  bacteria  and  leukocytes  without  serum — to 
detect  the  extent  of  spontaneous  phagocytosis. 

Readings. — A  great  number  of  fields  are  examined  microscopically, 
and  a  note  is  made  of  the  weakest  dilution  that  still  favors  phagocytosis. 


206  OPSONIC    INDEX 

No  attempt  is  made  to  count  the  phagocyted  bacteria.  The  relative 
amount  of  phagocytosis  with  the  immune  serum  in  various  dilutions  is 
compared  with  the  normal  controls,  and  the  result  is  expressed  as  the 
bacteriotropic  titer. 

Simon's  Method. — This  method  of  counting  the  number  of  empty 
leukocytes  with  a  given  dilution  of  serum  is  followed;  a  similar  count  is 
made  of  the  normal  serum  control  in  the  same  dilution;  thus,  if  in  the 
control  film  25  per  cent,  of  leukocytes  were  empty,  and  in  the  patient's 
film,  50  per  cent.,  the  index  would  be  |~f  =  0.5.  A  study  of  the  results 
obtained  by  this  method,  and  by  careful  counting  after  Wright's  method, 
shows  that  they  are  fairly  comparable,  and  the  method  may  be  used 
where  it  is  only  necessary  to  determine  whether  the  index  is  high  or 
low. 

Precautions. — 1.  If  phagocytosis  is  entirely  absent,  one  should  not 
conclude  that  bacteriotropins  are  not  present.  The  leukocytes  may 
have  been  injured,  especially  if  heterologous  leukocytes  have  been 
present;  control  examinations  with  homologous  leukocytes  (i.  e.,  from 
the  same  animal)  as  the  serum  should  result  in  phagocytosis. 

2.  The  time  during  which  the  tubes  were  in  the  incubator  may  have 
been  too  short  or  too  long.     Most  microorganisms  require  from  one- 
half  to  two  hours — meningococci  require  about  one-half  hour;  pneumo- 
cocci  usually  need  at  least  two  hours;  typhoid  and  cholera  bacilli  about 
fifteen  minutes  to  thirty  minutes,  as  they  undergo  extracellular  or  even 
intracellular  lysis  quite  readily. 

3.  If  the  control  of  bacteria  and  leukocytes  alone  shows  well-marked 
phagocytosis  the  test  should  be  repeated  with  another  strain. 

PRACTICAL  VALUE  OF  THE  OPSONIC  INDEX 

1.  In  competent  hands,  the  opsonic  index  of  normal  persons  to  most 
pathogenic  organisms  has  been  found  to  vary  from  0.8  to  1.2.     As 
previously  mentioned,  it  is  difficult  to  find  a  perfectly  normal  serum  for 
such  microorganisms  as  the  Bacillus  coli,  the  staphylococci,  Bacillus 
tuberculosis,  etc.,  as  it  is  unlikely  that  -any  individual  can  altogether 
escape  active  infection  at  some  period  of  his  life.     As  menstruation  ap- 
proaches, even  wider  fluctuations  occur,  the  normal  index  being  re- 
established by  the  second  or  third  day.     During  the  first  year  of  life  the 
opsonic  index  varies  to  such  a  degree  that  it  has  little  or  no  practical  value. 

2.  Although  a  large  amount  of  work  has  been  done  with  the  op- 
sonins  in  disease,  it  is  the  consensus  of  opinion  that  the  determination  of 


QUANTITATIVE    ESTIMATION    OF    BACTERIOTROPINS 


207 


the  opsonic  index  has  less  practical  significance  than  was  originally  be- 
lieved. One  point  is  clear,  however,  that  the  work  of  Wright  and  others 
has  broadened  the  field  of  vaccine  therapy  and  placed  it  upon  a  firmer 


Day  of 
Month 


AT 


/x" 


I I 


i 


J 


£±± 


FIG.  56. — AN  OPSONIC  INDEX  CHART. 

foundation.  Aside  from  the  10  per  cent,  of  chances  of  technical  error 
in  making  an  opsonin  measurement,  other  factors  may  be  present  that 
are  entirely  beyond  control  and  cannot  be  measured  by  the  immunisator, 
and  that  may  seriously  affect  the  value  of  the  opsonic  index. 

3.  As  a  diagnostic  procedure,  Wright  has  shown  that  the  opsonins 
possess  a  certain  specificity,  and  that  in  a  given  infection  a  low  index 


208  OPSONIC    INDEX 

for  a  certain  microorganism  indicates  that  this  organism  is  probably  the 
etiologic  factor.  This  possibility  is  strengthened  if  the  opsonic  index 
for  this  microorganism  is  increased  by  careful  manipulation  or  exercise 
of  the  diseased  part,  when  auto-inoculation  occurs,  with  consequent 
increase  of  opsonin. 

4.  In  prognosis  the  opsonic  index  may  have  some  value  in  deciding 
whether  an  infection  has  been  entirely  overcome  or  is  still  active.     An 
attempt  is  made  to  induce  auto-inoculation,  as  by  gentle  massage  of  a 
knee-joint  or  a  hip;    active  exercise;    deep  breathing,  etc.,  and  the  in- 
dex is  made  before,  and  at  frequent  intervals  after,  such  attempts.     If 
the  index  remains  unchanged  within  the  normal  limits,  the  assumption 
is  that  the  infection  has  been  overcome;    if,  on  the  other  hand,  an  in- 
crease in  opsonin  occurs,  this  indicates  that  an  active  focus  remains. 

5.  Most  value  was  placed  by  Wright  upon  the  opsonic  index  as  a 
guide  to  the  size  and  frequency  of  doses  of  bacterial  vaccines  in  the  treatment 
of  disease.     A  large  number  of  careful  determinations  showed  that  an 
injection  of  vaccine  is  followed  by  a  decrease  of  the  opsonins  (negative 
phase),  which  is  of  variable  degree  and  duration,  according  to  the  amount 
injected  (Fig.  55).     This  is  followed  by  an  increase  (positive  phase), 
and  coincidentally  there  is  a  corresponding  improvement  in  the  patient's 
condition.     This  subject  is  discussed  more  fully  in  the  chapter  on  Active 
Immunization. 

The  purpose  of  proper  vaccination,  therefore,  is  so  to  gage  and 
time  the  different  doses  that  a  pronounced  or  prolonged  negative  phase 
is  prevented,  as  far  as  possible,  and  a  high  positive  phase  secured  and 
maintained.  It  is  obvious  that  the  technic  of  opsonic  measurement  con- 
sumes much  time,  and  that  the  immunisator  cannot  mark  the  index  at 
the  time  a  dose  of  vaccine  is  given.  However,  the  determination  of  the 
opsonic  index  at  proper  intervals  after  the  first  dose  of  vaccine  may  give 
valuable  information  as  regards  the  reaction  of  the  patient,  and  serve 
as  a  guide  to  the  size  and  frequency  of  subsequent  doses. 

As  a  routine  measure,  the  opsonic  index  has  fallen  into  disuse,  vac- 
cine therapy  being  largely  guided  by  the  clinical  evidences  of  reaction 
and  the  condition  of  the  patient.  That  it  has  distinct  value,  particularly 
in  scientific  investigation,  is  generally  admitted,  and  it  is  well  to  re- 
member that  in  the  early  years  following  Wright's  investigations  the 
practice  of  vaccine  therapy  was  limited  to  those  skilled  in  determining 
the  index,  preparing  the  vaccine,  and  carefully  guiding  and  guarding 
its  administration.  It  is  to  be  regretted  that  the  wholesale  and  indis- 


QUANTITATIVE    ESTIMATION    OF    BACTERIOTROPINS  209 

criminate  manufacture  and  use  of  vaccines  have  brought  this  valuable 
field  of  therapy  inevitably  into  disrepute.  This  is  being  realized  more 
and  more,  and  the  effort  is  being  made  to  restore  the  value  of  this  form 
of  therapy.  This  effort  consists  in  recognizing  the  possibilities  and 
limitations  of  the  method,  and  confining  its  practice  to  those  who  possess, 
at  least,  sufficient  knowledge  of  bacteriology  to  prepare  a  vaccine  and 
make  an  opsonic  measurement,  the  best  results  being  secured  by  co- 
operation between  bacteriologist  and  clinician. 
14 


CHAPTER  XIII 
BACTERIAL  VACCINES 

IN  this  chapter  a  method  for  preparing  bacterial  vaccines  will  be 
described,  the  general  discussion  of  vaccine  therapy,  with  the  special 
technic  for  preparing  cow-pox  vaccine,  rabies  vaccine,  tuberculin,  and 
other  special  vaccines,  being  taken  up  in  Chapter  XXIX. 

Definition. — Bacterial  vaccines  are  "sterilized  and  enumerated  sus- 
pensions of  bacteria  which  furnish,  when  they  dissolve  in  the  body,  sub- 
stances which  stimulate  the  healthy  tissues  to  a  production  of  specific 
bacteriotropic  substances  which  fasten  upon  and  directly  or  indirectly  con- 
tribute to  the  destruction  of  the  corresponding  bacteria"  (Wright). 

TECHNIC  FOR  PREPARING  BACTERIAL  VACCINES 

Bacterial  vaccines  are  made — (1)  By  procuring  the  infected  ma- 
terial; (2)  by  preparing  pure  cultures  of  the  bacteria  that  are  to  be 
attacked;  (3)  by  making  suspensions  of  these  in  saline  solution,  adding 
a  preservative,  and  placing  in  proper  containers. 

1.  Procuring  Infected  Material. — Various  precautions,  according 
to  existing  circumstances,  should  be  taken  to  avoid  contamination  and 
to  secure  material  that  is  truly  representative  of  the  focal  secretions. 
For  instance,  pus  should  be  collected  from  an  abscess  cavity  or  sinus 
after  the  surrounding  tissues  have  been  cleansed  with  dilute  tincture  of 
iodin,  for  if  we  secured  a  culture  of  the  relatively  harmless  Staphy- 
lococcus  epidermidis  albus  from  the  skin  instead  of  the  Staphylococcus 
aureus,  which  may  be  the  cause  of  infection,  our  vaccine  will  have  little 
or  no  value. 

Nasal  secretion  may  be  secured  after  cleansing  the  nasal  orifice  with 
soap  and  warm  water,  passing  a  sterile  cotton  swab  through  a  nasal 
speculum,  and  rubbing  the  surfaces  of  the  lower  turbinates  and  septum 
lightly. 

An  ear  should  be  cleansed,  the  excess  of  secretions  removed  with 
sterile  swabs,  and  the  culture  be  made  of  pus  from  the  infected  tissues. 
Various  saprophytes  quickly  gain  admission  and  grow  in  the  necrotic  pus, 
whereas  the  infecting  bacterium  is  more  likely  to  be  found  in  the  tissues. 

210 


TECHNIC    FOR   PREPARING   BACTERIAL   VACCINES  211 

In  the  collection  of  sputum  special  care  is  required:  the  patient 
should  be  instructed  to  brush  the  teeth  with  a  sterilized  brush,  rinse  the 
mouth  several  times  with  boiled  water,  and  after  swallowing  several 
mouthfuls  of  water,  to  cough  and  expectorate  into  a  wide-mouthed 
sterilized  bottle.  The  sputum  may  be  plated  at  once,  or  washed  sev- 
eral times  in  sterile  Petri  dishes  with  sterile  water  and  then  cultured. 

Lung  Puncture  may  occasionally  be  required  in  infective  lung  con- 
ditions in  which  sputum  is  not  obtainable  or  is  too  badly  contaminated. 
A  5  to  10  c.c.  all-glass  syringe  with  a  strong  needle  is  sterilized  by  boiling, 
and  2  or  3  c.c.  of  peptone  broth  introduced.  The  skin  of  the  chest-wall 
over  the  site  of  infection,  as  shown  by  clinical  evidence,  is  sterilized  with 
tincture  of  iodin,  and  puncture  made  into  the  pulmonary  tissues.  When 
the  desired  depth  has  been  reached,  1  c.c.  of  the  broth  is  injected  gently 
into  the  tissues,  and  after  the  lapse  of  a  few  seconds  reaspirated  as  far 
as  possible  into  the  syringe.  During  the  operation  the  patient  should 
refrain  from  respiratory  movements,  in  order  to  minimize  any  risk  of 
lacerating  the  pulmonary  tissues  (Allen). 

Urine  should  always  be  withdrawn  with  a  sterile  catheter  after 
thoroughly  cleansing  the  meatus.  This  last  is  especially  important,  for 
the  infection  may  be  due  to  a  certain  strain  of  Bacillus  coli,  and  unless 
we  are  successful  in  obtaining  a  culture  of  this  particular  strain,  the 
vaccine  will  have  little  value. 

Blood  specimens  are  taken  with  a  sterile  syringe  from  a  prominent 
vein  at  the  elbow  after  the  skin  has  been  cleansed  and  sterilized  with 
tincture  of  iodin,  and  cultured  in  large  amounts  on  proper  culture-media. 

2.  Preparing  Pure  Cultures. — This  is  frequently  the  most  difficult 
step  in  the  whole  technic,  for  some  microorganisms,  as,  for  example,  the 
gonococcus  and  Bacillus  influenzse,  grow  slowly,  require  special  culture- 
media,  and  their  colonies  may  easily  be  overlooked.  To  secure  pure 
cultures,  and  especially  to  select  one  or  at  most  two  organisms  that  may 
be  the  chief  offenders,  considerable  bacteriologic  knowledge  is  necessary, 
and  no  simple  rules  or  directions  can  be  given  in  the  limited  space  of  this 
volume. 

1.  Stained  smears  of  the  secretions  of  a  lesion  may  indicate  the  nature 
of  the  infection  and  the  best  culture-medium  and  technic  to  use  for  pur- 
poses of  isolation. 

2.  Cultures  of  the  lesion  may  be  made  upon  solid  media,  and  isola- 
tion carried  out  after  a  primary  growth  has  been  secured.     With  proper 
care,  primary  cultures  may  be  grown,  such  as  staphylococci  from  the 
pus  of  a  freshly  incised  abscess,  or  the  microorganism  of  a  case  of  cystitis 


212  BACTERIAL   VACCINES 

or  pyelitis  by  securing  urine  with  the  aid  of  a  sterilized  catheter.  If 
slowly  growing  organisms,  such  as  Bacillus  influenzas,  gonococcus, 
pneumococcus,  etc.,  are  to  be  cultured,  "streak"  plates  are  usually  sat- 
isfactory, and  as  a  routine  the  best  culture-media  are,  as  a  rule,  those 
containing  serum  or  blood. 

3.  The  cultures  that  are  to  be  worked  up  into  a  vaccine  are  usually 
best  made  on  solid  media. 

4.  In  making  a  bacterial  vaccine  of  a  freely  growing  microorganism 
for  an  individual  patient,  it  will  suffice  to  plant  two  agar  slant  tubes; 
when  dealing  with  bacteria  that  grow  much  less  luxuriantly,  such  as 
streptococci  and  pneumococci,  four  to  six  tubes  should  be  used. 

5.  Cultures  are  usually  grown  for  twenty-four  hours  at  37°  C.;  but 


FIG.  57. — PREPARATION  OF  A  BACTERIAL  VACCINE. 

Removing  the  culture  of  bacteria  by  gently  rubbing  over  the  medium  (agar-agar) 
with  a  sterilized  platinum  loop. 

in  the  case  of  rapidly  growing  organisms  a  shorter  period  in  the  in- 
cubator will  suffice. 

6.  When  the  cultures  are  ready,  a  smear  of  each  growth  is  made  and 
stained  in  order  to  see  that  pure  cultures  of  the  right  microorganism  were 
made. 

7.  Inasmuch  as  the  immunizing  power  of  a  vaccine  is  in  most  cases 
a  factor  of  the  virulence  of  the  organism,  this  being  especially  true  of 
such    organisms    as    the  pneumococcus,  streptococcus,    and  influenza 
bacillus,  it  is  well,  whenever  possible,  to  employ  the  first  pure  subculture 
for  the  preparation  of  the  vaccine. 

3.  Preparation  of  the  Emulsion. — Carefully  observing  aseptic  pre- 
caution throughout,  pour  a  portion  of  a  test-tube  of  a  sterile  normal  salt 
solution  over  the  surface  of  the  first  culture,  shaking  the  fluid  in  such  a 


TECHNIC  FOR   PREPARING   BACTERIAL  VACCINES  213 

way  as  to  bring  the  microorganisms  into  suspension.  If  the  culture  is 
not  easily  washed  from  the  medium,  a  sterile  platinum  loop  may  be  used 
to  remove  the  growth,  care  being  taken  not  to  cut  into  the  medium  and 
mix  the  fragments  with  the  bacterial  suspension  (Fig.  57). 

The  bacterial  suspension  thus  obtained  is  poured  on  the  surface  of 
the  second  culture,  bringing  this  into  suspension,  and  repeating  the 
process  until  the  whole  series  of  cultures  have  been  suspended,  adding 
more  salt  solution  if  necessary. 

The  final  suspension  is  transferred  to  a  sterile,  thick-walled  flask 
containing  glass  beads,  and  shaken  by  hand  or  in  a  mechanical  shaker 
until  the  bacterial  masses  are  broken  up  (Fig.  58).  This  may  be  es- 
pecially difficult  with  diphtheria  bacilli  and  streptococci.  Unless  the 


FIG.  58. — A  SHAKING  APPARATUS  (Electric,  110  volts,  direct). 
A  satisfactory  machine  for  shaking  vaccines,  preparing  antigens,  making  emulsions, 

etc. 

emulsion  is  perfectly  homogeneous,  the  larger  particles  may  be  removed 
by  brief  centrifugalization  or,  better,  by  filtering  through  a  sterile  filter. 
There  is  evidence  to  show  that  bacteria  grown  on  culture-media  con- 
taining peptone  may  produce  objectionable  toxic  substances  capable 
of  producing  anaphylactic  phenomena  (Reichel  and  Harkins1).  In  addi- 
tion, when,  in  the  preparation  of  a  vaccine,  bacteria  grown  on  a  serum 
medium  are  washed  off  with  normal  salt  solution,  a  portion  of  the  serum 
may  be  removed  and  this  may  be  capable  of  producing  disagreeable 
local  and  general  reactions.  For  these  reasons  it  is  advisable  to  wash  all 
suspensions  by  repeated  centrifugalization  until  the  supernatant  fluid 
reacts  negatively  to  the  biuret  or  ninhydrin  reaction  (Willard  Stone). 

1  Centralb.  f.  Bakteriolog.,  etc.,  1913,  69. 


214 


BACTERIAL   VACCINES 


4.  Counting  of  the  Bacterial  Suspension. — Standardization,  best  ac- 
complished by  counting  the  bacterial  elements  con- 
.g         tained  in  a  unit  volume  of  the  suspension,  is  necessary 
in  order  to  adjust  our  initial  dose  as  experience  will 
*"§         dictate  and  for  guidance  in  making  subsequent  in- 
jections. 

•g  In  dealing  with  a  vaccine  we  have  to  count  both 

the  dead  and  the  living  bacteria,  making  no  dis- 
tinction, for  both  furnish  the  chemical  agent  that 
calls  forth  the  elaboration  of  bacteriotropic  sub- 
stances. Inasmuch  as  sharp  definition  and  the 

|  ^         staining  properties  of  bacteria  may  be  lost  in  the 
process   of   sterilization   by  heat,  the   specimen  of 

3  1         vaccine  to  be  examined  should  be  secured  before 

|  -§         sterilization  is  undertaken. 

pq  -3  The   counting  or  standardization  may  be  done 

•<  |         in  several  ways:    (a)  By  mixing  equal  portions  of 
normal  blood  and  bacterial  emulsion  and  counting 

g  «g\      the  proportion  of  corpuscles  to  bacteria  in  our  mix- 

O  |T      ture  (Wright) ;  (6)  by  diluting,  staining,  and  counting 

0  ,|  &      with  the   hemo   cytometer,  as  in  the  enumeration 
H  ^  2      of  red  blood-corpuscles;   (c)  for  standardizing  large 

^jU    &  g,         quantities  of  bacterial  vaccine  the  method  of  Kolle 
.  tj10    (x  g         or  (d)  that  of  Hopkins  may  be  used. 
*§      5  "5  (°0  Method  of  Wright. — Prepare  a  simple  capil- 

S  ;§         lary  pipet,  making  a  mark  on  its   stem  about  an 

*  fe 

•-     °  —         inch  from  the  tip,  and  fit  a  teat  to  its  barrel  (Fig. 

<(j     C^ 

1  *§         59).     Cleanse  and  prick  the  finger,  press  out  a  drop 
S  ~         of  blood,  take  up  the  pipet  and  draw  up  into  it  first 

"g      g  .g         one  volume  of  sodium  citrate  solution,  one  of  blood 
j§     ^  g         and  then  either  one  volume  of  bacterial  suspension 

*N  tja 

I         or  two  or  more  volumes,  if  it  appears  on  inspection 
to  contain  much  fewer  than  500,000,000  of  bacteria 

•b  m 

g         to  the  cubic  centimeter.     To  guard  against  crimping 
|  of  the  corpuscles  in  drying  the  films,  Wright  advo- 

!§  |      •  "f         cates  aspirating  one  or  two  volumes  of  distilled  water 
^       ^         after  the  blood  and  bacterial  suspension, 
fl?         |3  Now  expel  from  the  pipet  first  only  the  distilled 

water  and  bacterial  emulsion,  and  mix  these,  so  that 
there  may  be  no  danger  of  the  red  corpuscles  becoming  hemolyzed,  and 


-  •  , 


FIG.   60. — A  SATISFACTORY  PREPARATION  FOR  COUNTING  A  BACTERIAL  VACCINE. 
The  microorganisms  are  well  separated  and  evenly  distributed  among;  the  cor- 
puscles;   the  spread  is  even  and  regular,  and  the  corpuscles  are  not  gathered  in 
rouleau  formation  or  irregular  clumps. 


FIG.  61. — AN  UNSATISFACTORY  PREPARATION  FOR  COUNTING  A  BACTERIAL  VACCINE. 
The  microorganisms  are  mostly  gathered  in  irregular  masses  instead  of  being 
separated  and  evenly  distributed;  the  corpuscles  are  not  separated  and  evenly  dis- 
tributed, but  show  a  tendency  to  gather  in  clumps;  both  factors  render  a  count 
difficult,  inaccurate,  and  unsatisfactory 


TECHNIC   FOR   PREPARING   BACTERIAL   VACCINES  215 

then  proceed  to  mix  together  the  whole  contents  of  the  pipet,  aspirating 
and  reexpelling  these  a  dozen  times.  Then  make  two  or  three  micro- 
scopic films  from  the  mixture,  spreading  these  out  on  slides  that  have 
been  roughened  with  emery. 

The  films  are  dried  in  the  air,  fixed  by  immersing  them  for  two  min- 
utes in  a  saturated  solution  of  corrosive  sublimate,  washed  thoroughly, 
and  stained  for  a  minute  with  carbolfuchsin  diluted  1 : 10  or  carbolthionin 
for  two  to  five  minutes,  and  then  washed  and  dried. 

The  films  are  now  given  a  preliminary  examination.  If  red  cor- 
puscles and  bacteria  are  found  in  approximately  the  same  numbers  and 
the  suspension  is  free  from  bacterial  aggregates,  the  count  may  be  made 
(Fig.  60).  If  either  the  bacteria  or  the  corpuscles  are  largely  in  excess, 
new  mixtures  and  new  films  must  be  made.  In  case  the  bacteria  are 
gathered  in  clumps,  the  suspension  should  be  shaken  again  and  new 
films  prepared  (Fig.  61). 

When  satisfactory  films  have  been  obtained,  the  actual  counting 
may  be  done.  This  is  carried  out  with  an  oil-immersion  lens,  and  in 
order  to  secure  accuracy,  it  is  necessary  to  restrict  or  divide  the  field  by 
a  small  square  diaphragm  made  of  paper  or  cardboard,  or  by  inscribing 
cross  lines  on  a  small  clean  cover-glass  and  dropping  them  on  the  dia- 
phragm of  the  eye-piece. 

A  field  is  now  chosen  at  random,  and  the  corpuscles  and  bacteria 
are  counted,  the  results  being  jotted  down  on  a  sheet  of  paper,  keeping 
each  enumeration  separate  and  writing  the  numbers  in  two  columns. 
Proceed  at  random  from  field  to  field,  traversing  every  part  of  the 
slide.  Establish  a  rule  for  counting  corpuscles  that  transgress  or  touch 
the  edge  of  the  field.  Eliminate  from  consideration  any  parts  of  the 
films  in  which  the  preparation  is  unsatisfactory  as  regards  the  staining, 
or  with  respect  to  the  integrity  of  the  red  corpuscles.  The  examination 
is  continued  until  at  least  500  corpuscles  have  been  counted,  half  of  the 
count  being  made  from  the  second  slide.  The  number  of  microorganisms 
counted  is  now  totaled,  and  the  approximate  number  per  cubic  centi- 
meter estimated.  Let  us  assume,  for  example,  that  600  red  cells  and 
1200  bacteria  have  been  counted.  Now,  a  cubic  millimeter  of  blood 
contains  5,500,000  red  corpuscles,  and  equal  volumes  of  blood  and  emul- 
sion were  taken.  A  cubic  millimeter  of  the  emulsion,  therefore,  contains 

—  =  11,000,00,0    organisms     per    cubic     millimeter,    or 
600 

11,000,000,000  per  cubic  centimeter. 

(6)     The  second  method  of  counting  is  precisely  similar  to  that  em- 


216; 


BACTERIAL   VACCINES 


ployed  for  the  enumeration  of  blood-corpuscles,  the  diluting  and  staining 
fluid  being  made  by  adding  to  a  1  per  cent,  solution  of  sodium  chlorid 
in  distilled  water  sufficient  formalin  to  make  2  per  cent.,  and  alcoholic 
gentian-violet,  5  per  cent.  The  emulsion  is  drawn  up  in  a  white  cor- 
puscle pipet  to  the  mark  0.5,  and  with  diluting  fluid  to  the  mark  11. 
The  contents  are  then  mixed  thoroughly  for  several  minutes,  several 
drops  expelled,  a  drop  placed  in  the  counting  chamber  and  properly 
covered  with  a  special  thin  cover-glass.  The  bacteria  are  allowed  to 

remain  in  the  counting  cell  for 
at  least  half  an  hour  prior  to 
enumeration.  A  large  number 
of  small  squares  are  counted, 
and  the  average  of  one  square 
obtained.  By  multiplying  this 
figure  by  4000  and  then  by  20, 
the  number  of  bacteria  per 
cubic  millimeter  is  obtained, 
and  1000  times  this  figure 
gives  the  number  of  bacteria 
contained  in  one  cubic  centi- 
meter of  the  vaccine.  If  the 
emulsion  is  highly  concen- 
trated, the  red  cell  pipet  may 
be  used  and  the  calculations 
made  accordingly. 

(c)     Method  of  Kolle.—A 

FIG.62.-INSTRUMENTFORTHESTANDARDIZA-      P^Orm    1<X>P    adjusted    to    fit 

TION  OF  PLATINUM  LOOPS.  No.  2  of  a  Lautenschlager  wire 


4  mm.,  and  holds  approxi- 
mately 2  mg.,  or  about  2,500,000,000  organisms.  By  growing  an 
organism  on  slants  of  agar  and  emulsifying  a  certain  number  of  loopf  uls 
in  a  measured  quantity  of  saline  solution,  an  approximate  method 
of  standardization  is  obtained.  According  to  Kolle,  ordinary  slants  of 
agar  will  hold  about  15  loopf  uls  of  staphylococci,  Bacillus  typhosus, 
or  Bacillus  coli,  and  about  5  loopfuls  of  streptococci  and  gonococci. 

(d)     Method  of  Hopkins.* — This   is   based    upon   concentrating  a 
bacterial  suspension  by  centrif ugalization  and  the  preparation  of  stand- 
ard emulsions  from  the  sediment.    The  emulsion  is  filtered  through  a 
1  Jour.  Amer.  Med.  Assoc.,  1913,  xl,  1615. 


TECHNIC   FOR   PREPARING   BACTERIAL   VACCINES 


217 


small  cotton  filter  to  remove  larger  clump  of  bacteria  and  particles  of 
agar,  and  is  then  placed  in  specially  constructed  centrifuge  tubes  (Inter- 
national Centrifuge  Company,  see  Fig.  63),  covered  with  rubber  caps, 
and  centrifugal ized  for  half  an  hour  at  a  speed  of  approximately  2,800 
revolutions  a  minute.  The  salt  solution  and  bacteria  above  the  0.05 
mark  are  then  removed,  and  5  c.c.  saline  solution  is  measured  into  the 
tube,  so  as  to  make  a  1  per  cent,  emulsion.  If  the  sediment  does  not 
reach  the  0.05  mark,  its  volume  is  read  on  the  scale,  and  a  corresponding 
quantity  of  saline  is  added  to  make  the  emulsion  1  per  cent,  in  strength. 
The  bacteria  are  forced  into  suspension,  the 
vaccine  transferred  to  a  sterile  tube,  and  the 
microorganisms  killed  in  the  usual  manner. 

Estimations  of  carefully  counted  suspensions 
obtained  by  centrifugalization  in  the  foregoing 
manner  gave  the  following  results : 


PER 

CENT 


Staphylococcus  aureus  and  albus 

Streptococcus  hsemolyticus 

Gonococcus 

Pneumococcus 

Bacillus  typhosus 

Bacillus  coli .  . 


BILLION 
PER  C.C. 

10.0 
8.0 
8.0 
2.5 
8.0 
4.0 


5.  Sterilizing  the  Vaccine  and  Testing  its 
Sterility. — When,  after  the  preliminary  examina- 
tion, the  films  for  counting  have  been  found  satis- 
factory, a  pause  is  made  to  start  the  process  of 
sterilization,  which  may  continue  while  the  count 
is  being  made.  Either  heat  or  a  germicide  may  be 
used  for  sterilizing  vaccine,  preferably  the  former. 

The  vaccine  may  be  placed  in  a  test-tube, 
which  is  then  sealed  (Fig.  64),  and  the  whole  im- 
mersed in  the  water-bath;  a  simpler  method,  and 
one  just  as  good,  is  to  place  the  flask  or  tube  of 

vaccine  in  the  bath,  observing  special  care  to  see  that  the  water  is  above 
the  level  of  the  vaccine. 

Efficient  sterilization  is  dependent  upon  permitting  the  process  to  con- 
tinue at  the  minimum  temperature  for  the  minimum  length  of  time.  With 
the  water-bath  at  56°  to  60°  C.  sterilization  is  nearly  always  complete  in 
an  hour. 

The  vaccine  should  be  now  cultured  to  test  its  sterility.  At  least  a 
dozen  platinum  loopfuls  are  transferred,  under  strict  aseptic  precautions, 


FIG.  63.  —  HOPKINS' 
TUBE  FOE  STAND- 
ARDIZING A  BACTE- 
RIAL VACCINE. 


218 


BACTERIAL   VACCINES 


to  a  slant  of  a  suitable  culture-medium,  such  as  Loffler's  blood-serum  or 
blood-agar;  this  is  incubated  at  least  twenty-four  hours,  or  longer  if  the 
organism  is  a  slowly  growing  one.  It  is  then  examined,  and  if  found 
sterile,  the  preparation  of  the  vaccine  may  be  completed.  If  not,  the 
vaccine  is  heated  for  another  hour,  or,  preferably,  a  new  vaccine  is 
prepared. 

6.  Diluting  the  Vaccine  and  Adding  a  Preservative. — Having  made 
the  count  and  sterilized  the  vaccine,  it  is  next  diluted  with  sterile  saline 

solution  so  that  each  cubic  centimeter 
contains  the  dose  decided  upon.     If 


FIG.  64A. — A  STOCK  AMPULE  OF 
VACCINE. 


FIG.  64B. — STOCK  BOTTLE  OF  BACTERIAL 
VACCINE. 


the  treatment  is  likely  to  be  prolonged,  a  sufficient  number  of  doses 
should  be  provided  for.  It  is  a  good  plan  not  to  dilute  all  the  vaccine, 
but  to  preserve  the  remainder  undiluted  in  case  larger  doses  are  subse- 
quently needed.  If,  for  instance,  a  vaccine  of  Staphylococcus  aureus 
contains  1,500,000,000  organisms  per  cubic  centimeter  and  the  dose 
decided  upon  is  500,000,000  per  cubic  centimeter,  sufficient  vaccine  for 
30  doses  is  prepared  by  withdrawing  10  c.c.  of  vaccine  in  a  sterile  con- 
tainer and  adding  20  c.c.  of  sterile  salt  solution.  This  mixture  is  agitated, 


TECHNIC   FOR   PREPARING   BACTERIAL   VACCINES 


219 


to  insure  thorough  mixing,  and  01  c.c.  of  a  1:1 00 
added  to  each  cubic  centimeter  of  vaccine  as  a 
preservative  against  chance  contamination.  Thus, 
in  the  foregoing  example,  3  c.c.  of  the  diluted 
phenol  would  be  added.  The  amount  of  stock 
vaccine  is  estimated  or  measured,  0.5  per  cent, 
phenol  is  added,  and  the  vaccine  stored  in  a 
sterile  container  in  the  refrigerator,  being  first 
properly  labeled  with  the  patient's  name,  the 
date,  and  the  number  of  bacteria  per  cubic  centi- 
meter. 

The  vaccine  may  now  be  placed  in  a  sterile 
vaccine  bottle,  fitted  with  a  sterile  rubber  cap, 
and  properly  labeled  (Fig.  64).  When  it  is  to  be 
administered,  the  cap  is 
touched  with  tincture  of  iodin, 
the  needle  plunged  through  the 
cap,  and  a  dose  withdrawn  with 
a  sterile  syringe.  The  punc- 
ture in  the  cap  is  then  sealed 
with  a  drop  of  flexible  collo- 
dion. This  method  is  inex- 
pensive, and  with  proper  care 
is  quite  satisfactory,  especially 
for  stock  vaccines. 

Another  and  probably 
better  method,  especially  for 
autogenous  vaccines,  consists 
in  tubing  each  dose  in  separate 
sterile  ampules  (Fig.  65),  which 
are  then  sealed  in  the  flame.  When  the  vaccine 
is  fco  be  administered,  the  ampule  is  well  shaken, 
the  neck  broken  in  a  towel,  and  the  contents 
aspirated  into  a  sterile  syringe.  These  ampules 
may  be  purchased  ready  for  use  or  be  made  in 
the  laboratory,  using  6  mm.  soft  glass  tubing 
(Fig.  6).  For  pipeting  a  vaccine  into  ampules  the 
special  automatic  pipet  shown  in  the  illustration 
(Fig.  66)  is  quite  convenient.  As  a  rule,  vac- 
cines should  be  preserved  in  a  cool  place,  such  as 


dilution  of  phenol  is 

mmm 


FIG.  65. —  A  SMALL 

VACCINE  AMPULE. 

Capacity  1  c.c. 


FIG.  66. —  COMER'S 
AUTOMATIC  PIPET. 
(Steele  Glass  Co., 
Philadelphia.) 

The  inner  tube  to 
the  tip  of  the  pipet 
holds  exactly  1  c.c.  If 
the  rubber  teat  raises 
too  much  fluid,  the 
excess  is  received  in  the 
glass  reservoir;  when 
too  much  fluid  accumu- 
lates in  this,  it  may  be 
emptied  by  turning  the 
point  of  the  inner  tube 
downward  and  ejecting 
the  fluid  by  pressure  on 
the  teat. 


a  refrigerator. 


220  BACTERIAL   VACCINES 

PREPARATION  OF  SENSITIZED  BACTERIAL  VACCINES 

A  highly  immune  serum  is  prepared  by  immunizing  a  series  of  rabbits 
or  a  goat  with  the  microorganism  to  be  used  in  preparing  the  vaccine. 
The  first  injections  consist  of  heat-killed  emulsions,  administered 
subcutaneously.  After  the  first  or  second  dose  the  period  of  heating  is 
gradually  reduced,  and  the  dose  increased,  until  finally  the  injections 
may  be  given  intravenously  and  with  living  microorganisms.  From  time 
to  time  a  small  amount  of  serum  should  be  examined  for  immune 
bodies :  with  the  typhoid-cholera  group,  by  testing  for  bacteriolysin  and 
agglutinins;  with  staphylococci  and  streptococci,  by  agglutination, 
bacteriotropic,  and  complement-fixation  tests;  with  pneumococci,  gon- 
ococci,  and  meningococci,  by  bacteriolytic,  agglutination,  and  bacterio- 
tropic tests.  When  a  highly  immune  serum  is  secured,  the  animal  is 
bled,  the  serum  isolated,  heated  to  56°  C.  for  half  an  hour,  and  stored 
in  a  strictly  aseptic  manner. 

To  "sensitize"  the  bacteria,  thick,  even  emulsions  of  young  cultures 
in  normal  salt  solution  are  treated  with  one-half  to  an  equal  bulk  of 
inactivated  immune  serum,  and  the  mixture  gently  agitated  at  room 
temperature  for  from  six  to  twelve  hours.  The  emulsion  is  then  thor- 
oughly centrifuged,  and  the  residue  of  bacteria  washed  three  times  with 
sterile  salt  solution,  after  the  manner  in  which  the  red  corpuscles  are 
washed.  After  the  final  washing  the  bacteria  are  resuspended  in  salt 
solution,  shaken  for  a  time  to  insure  breaking  up  of  agglutinated  clumps, 
counted,  heated  at  60°  C.  for  an  hour,  cultured  as  a  test  for  sterility,  and 
then  diluted  so  that  the  emulsion  will  contain  slightly  larger  doses  than 
a  corresponding  dose  of  ordinary  vaccine  prepared  for  administration. 

"  Sensitization "  probably  consists  in  the  union  of  bacteriolytic 
amboceptor  with  its  antigen,  and  when  injected,  serves,  with  the 
patient's  complement,  to  hasten  solution  or  lysis  of  the  bacteria  (antigen), 
thereby  liberating  quickly  the  chemical  substances  required  for  the 
stimulation  of  antibodies. 

Metchnikoff  and  Besredka  are  using  sensitized  living  bacteria,  and 
their  work  is  being  followed  with  much  interest.  In  this  country  strict 
legal  restrictions  and  regulations  exist  regarding  the  sending  of  living 
cultures  through  the  mails.  If,  therefore,  the  method  should  fulfil  the 
high  claims  and  expectations  made  for  it,  there  may  be  considerable 
difficulty  in  bringing  it  into  general  use. 


THE   ADMINISTRATION   OF  A   BACTERIAL   VACCINE  221 

THE  ADMINISTRATION  OF  A  BACTERIAL  VACCINE 

Syringe. — Vaccines  are  best  administered  with  the  aid  of  a  1  c.c. 
all-glass  syringe,  furnished  with  a  sharp  platinum  iridium  or  steel  needle. 
These  may  be  sterilized  in  boiling  water  for  a  minute  or  longer.  After 
sterilization,  the  parts  should  be  carefully  adjusted  and  the  syringe  loaded. 

According  to  Wright,  time  and  trouble  may  be  saved  by  sterilizing  in 
oil  at  a  temperature  of  125°  to  140°  C.  If  this  temperature  is  not  ex- 
ceeded, the  oil  can  be  drawn  into  the  syringe  without  danger  of  breaking 
the  glass.  The  procedure  is  as  follows:  Partly  fill  a  tablespoon  with 
any  vegetable  oil,  and  into  this  place  a  bread-crumb  about  the  size  of 
a  large  hemp-seed.  Then  heat  over  a  spirit-lamp  until  bubbles  of  steam 
begin  to  appear  about  the  bread-crumb.  The  temperature  of  the  oil 
is  now  about  that  of  boiling  water.  After  bubbles  have  ceased  to  form 
about  the  bread-crumb — meanwhile  drawing  up  the  oil  once  or  twice 
into  the  syringe — reapply  the  heat  very  cautiously  until  the  bread- 
crumb shows  the  first  sign  of  turning  brown  (at  about  140°  C.).  Then, 
without  allowing  time  for  the  oil  to  cool,  draw  it  up  two  or  three  times 
into  the  syringe,  being  careful  to  see  that  it  comes  into  contact  with 
every  part  of  the  interior. 

If  it  is  desired,  so  as  to  improve  the  appearance,  to  get  rid  of  all  re- 
maining traces  of  oil  from  the  syringe,  this  can  be  easily  effected  by 
drawing  up  into  the  syringe  a  very  weak  (0.25  to  0.50  per  cent.)  solution 
of  sterilized  sodium  carbonate. 

Method  of  Making  the  Inoculation. — As  the  administration  of  a 
vaccine  is  frequently  followed  by  a  temporary  depression  of  the  resist- 
ing powers  of  the  individual  and  a  feeling  of  lassitude,  the  injections  are, 
as  a  rule,  best  given  during  the  afternoon  and  evening,  the  night's  rest 
aiding  in  overcoming  the  depression.  Since  the  determination  of  proper 
dosage  rests  mainly  on  the  observation  of  such  clinical  signs  and  symp- 
toms as  temperature,  pulse,  and  local  reaction  at  the  site  of  the  lesion, 
the  patient  should  be  watched  during  the  following  twenty-four  to 
forty-eight  hours. 

The  injections  should  be  given  at  a  point  where  the  tissues  are  loose, 
where  muscular  action  is  not  much  in  evidence,  and  where  pressure  by 
clothing  or  weight  is  not  made.  The  most  suitable  localities  are  in 
front  of  the  shoulder,  at  a  site  about  1J^  inches  below  the  center  of  the 
clavicle;  high  up  in  the  buttock,  or  in  the  side  of  the  abdomen,  about 
two  or  three  inches  inside  the  anterior-superior  spine  of  the  ilium.  The 
skin  at  the  point  of  injection  should  be  touched  with  tincture  of  iodin, 


222  BACTERIAL   VACCINES 

which  is  removed  after  the  injection  has  been  given  by  washing  with 
alcohol. 

Both  convenience  and  experimental  work  to  test  the  comparative 
efficacy  of  inoculation  into  different  tissues  point  to  the  subcutaneous 
tissues  as  the  most  suitable  site  for  inoculation.  The  best  method  of 
procedure  is  to  pick  up  a  fold  of  skin  between  the  finger  and  thumb,  and 
then  to  push  the  needle  well  down  into  the  middle  of  the  fold,  and  slowly 
inject  the  fluid. 

Since  it  is  known  that  the  power  of  response  of  the  tissues  to  the 
stimulus  of  a  vaccine  is  somewhat  limited,  it  would  seem  advisable  to 
choose  a  new  site  for  each  successive  inoculation. 

The  Effects  of  Inoculation. — The  local  effects  produced  at  the  site 
of  inoculation  vary  considerably,  being  influenced  by  the  nature  of  the 
individual,  the  variety  and  amount  of  the  inoculum,  and  the  sensitive- 
ness of  the  patient's  tissues  to  stimulation.  In  the  majority  of  cases 
the  local  reaction  is  limited  to  a  very  slight  reddening  of  the  skin  around 
the  puncture  for  an  area  of  about  one  inch.  In  some  instances,  oc- 
casionally encountered  where  a  large  number  of  typhoid  inoculations 
have  been  made,  the  reaction  after  the  first  dose  is  more  severe  than 
after  subsequent  doses,  and  is  accompanied  by  considerable  edema, 
hyperemia,  and  pain. 

The  focal  effects  about  the  lesion  are  exceedingly  important  in  de- 
termining the  reaction  of  the  patient,  and  serve  as  a  guide  to  the 
adjustment  of  dosage  and  intervals.  Where,  in  a  case  of  furuncle,  an 
appropriate  dose  of  staphylococcus  vaccine  is  administered,  within  a 
few  hours  increased  hyperemia  is  seen  around  the  focus,  and  there  is  a 
slight  increase  in  the  swelling.  When  very  small  doses  are  given,  these 
focal  symptoms  may  practically  be  absent,  but,  as  a  rule,  a  slight  re- 
action does  no  harm,  but  serves  rather  to  show  that  the  vaccine  possesses 
some  degree  of  potency  and  may  aid  in  the  curative  process. 

The  constitutional  effects  may  also  vary  within  wide  limits.  An  ade- 
quate, but  not  excessive,  dose  may,  within  a  few  hours,  produce  a  feeling 
of  lassitude,  headache,  slight  rise  in  temperature,  and  acceleration  of  the 
pulse-rate.  Severe  constitutional  reactions  are  generally  due  to  ex- 
cessive dosage,  but  may  occur  in  some  persons  after  doses  that  were 
previously  well  borne. 

Frequency  and  Dosage  of  Inoculation. — No  definite  rules  can  be 
laid  down,  each  patient  being  a  law  unto  himself.  The  opsonic  index 
has  been  largely  abandoned  as  a  guide  to  the  administration  of  vaccine, 
the  reaction  and  condition  of  the  patient  now  governing  the  dosage. 


THE   ADMINISTRATION    OF   A   BACTERIAL   VACCINE  223 

In  more  acute  infections,  and  in  delicate  persons,  smaller  doses  are 
usually  indicated.  It  is  well  to  make  the  first  dose  small,  and  if  no  reac- 
tion occurs  within  forty-eight  hours,  a  second  and  a  larger  dose  may  be 
given.  If,  however,  the  patient  presents  other  symptoms  of  a  general 
reaction,  the  dose  given  was  large  enough,  and  may  be  repeated,  as 
necessary,  at  intervals  of  from  five  to  seven  days.  It  should  be  care- 
fully borne  in  mind  that  an  increase  in  dosage  is  contraindicated  so  long 
as  any  sign  of  general  or  focal  reaction  is  produced  and  steady  progress 
is  maintained.  One  should  always  be  on  guard  to  detect  any  signs  of 
fresh  infection  by  some  other  organism,  and  if  a  given  vaccine  is  failing 
to  exert  a  beneficial  effect,  additional  cultures  should  be  made,  instead 
of  continuing  to  administer  dose  after  dose  of  the  same  vaccine. 

The  intervals  at  which  injections  are  to  be  made  are  of  some  im- 
portance. It  is  certainly  better  to  wait  too  long  than  to  inoculate 
prematurely,  but  the  ghost  of  the  "negative  phase"  is  always  too  prom- 
inent in  the  minds  of  the  inexperienced.  The  inoculations  may  be 
given  while  improvement  is  still  in  progress  or  convalescence  well  es- 
tablished, in  the  endeavor  to  secure  a  summation  of  positive  phases  of 
clinical  improvement;  or  one  may  wait  for  the  first  signs  of  retrogression 
before  administering  another  dose.  The  former  method  is  the  preferable 
procedure,  but  is  difficult  to  accomplish;  the  latter  is  less  ideal,  but 
is  easier  to  perform  and  more  devoid  of  risk. 

The  dosage  varies  according  to  whether  the  infection  is  acute  or 
chronic,  the  nature  of  the  microorganism,  and  the  age  of  the  patient. 
No  fixed  rules  can  be  given.  In  acute  infections  the  dose  should  be 
small  and  may  frequently  be  repeated;  in  chronic  infections  larger  doses 
may  be  given  at  longer  intervals.  If  in  doubt  as  to  the  size  of  the  dose 
to  be  given,  it  is  better  to  give  a  small  dose,  and  carefully  observe  the 
effect  on  the  patient,  letting  this  serve  as  an  index  to  subsequent  doses. 
Children  tolerate  relatively  large  doses  of  bacterial  vaccines,  but  the 
dosage  should  depend  on  the  weight  and  not  on  the  age  of  the  child. 

The  following  is  a  list  of  the  ordinary  doses  for  adults  of  various  bac- 
terins : 

Staphylococcus  aureus 100,000,000  to  1,000,000,000 

Staphylococcus  albus  and  citreus 200,000,000  to  1,000,000,000 

Streptococcus  pyogenes 25,000,000  to     200,000,000 

Gonococcus 25,000,000  to     200,000,000 

Typhoid  bacillus 250,000,000  to  1,000,000,000 

Colon  bacillus 100,000,000  to  1,000,000,000 


CHAPTER  XIV 
ANTITOXINS 

FOR  general  purposes  the  antibodies  produced  during  infection  may 
be  divided  into  two  groups,  the  first  consisting  of  those  antibodies  that 
are  truly  antagonistic  to  the  bacterium  or  its  products  responsible  for 
their  production,  and  the  second  those  that  are  not  in  themselves  de- 
structive, but  that  probably  prepare  the  bacterium  for  the  action  of  a 
more  powerful  antibody  of  the  first  group. 

To  the  first  group  belong  the  antitoxins,  which  neutralize  the  toxins 
of  a  bacterium  without  being  directly  destructive  to  the  microorganism 
itself;  and  the  bacteriolysins,  which  are  truly  destructive,  causing  the 
bacterium  to  break  up  and  finally  disappear. 

To  the  second  group  belong  the  opsonins,  which,  as  we  have  seen, 
prepare  the  bacterium  for  phagocytosis;  and  the  agglutinins  and  pre- 
cipitins,  which,  while  not  in  themselves  destructive,  probably  in  some 
manner  prepare  their  antigen  for  the  action  of  bacteriolysins,  just  as 
opsonins  prepare  them  for  phagocytosis. 

Definition. — Antitoxins  are  antibodies  in  the  blood  that  are  capable  of 
directly  and  specifically  neutralizing  the  dissolved  toxins  that  caused  their 
production. 

Historic. — Bacteriolysins  were  discovered  before  antitoxins.  Their 
discovery  is  due  to  the  researches  of  Nuttall,  Fodor,  Buchner,  and  others, 
who  showed  that  normal  serum,  and  especially  the  serum  of  animals 
artificially  immunized  against  a  certain  bacterium,  was  able  to  exert  a 
destructive  action  on  the  microorganism,  causing  its  dissolution  and 
final  disappearance.  This  property  of  the  blood-serum  was  found  to 
diminish  with  age,  and  to  disappear  completely  when  the  serum  was 
heated  to  56°  C.  Buchner  laid  greatest  stress  upon  the  importance  of 
the  thermolabile  substance  which  he  called  alexin,  but  later  researches 
have  shown  that  the  main  factors  are  the  specific  bacteriolysins,  which, 
however,  are  practically  powerless  to  destroy  their  antigen  without  the 
cooperation  of  alexin  (later  renamed  " complement"  by  Ehrlich). 

While  these  studies  were  being  made,  in  the  hope  of  thus  explaining 
all  phases  of  immunity,  Behring  discovered  that  in  diphtheria  infections 

224 


FORMATION   OF   ANTITOXINS  225 

induced  experimentally,  while  the  animals  became  more  and  more  im- 
mune, virulent  bacilli  may,  nevertheless,  be  present  at  the  site  of  in- 
jection. Here,  then,  was  an  example  of  immunity  that  could  not  be 
explained  on  the  basis  of  bacteriolysis.  Later,  in  1890  and  1892,  Behr- 
ing,  in  collaboration  with  Kitasato  and  Wernicke,  made  further  im- 
portant discoveries,  showing  that  the  blood-serum  of  animals  actively 
immunized  against  diphtheria  and  tetanus  would  protect  normal  animals 
against  these  diseases,  and,  furthermore,  that  the  blood-serum  of  the 
immune  animals  did  not  possess  bactericidal  properties.  These  ob- 
servers also  demonstrated  that  such  serum  could  be  used  therapeutically 
for  the  cure  of  an  infection  already  in  progress. 

Soon  after  these  discoveries  Ehrlich  showed  that  specific  antitoxins 
(antiricin,  antiabrin,  etc.)  could  also  be  produced  against  the  poisons 
of  some  plants,  and  Calmette  produced  a  similar  antitoxin  (antivenin) 
against  snake  poison.  Other  observers  since  then  have  increased  the  list 
of  poisons  against  which  antitoxins  can  be  produced;  as,  for  example, 
Kempner  has  produced  an  antitoxin  against  the  poison  of  Bacillus  botu- 
linus,  and  Wassermann  one  against  that  of  Bacillus  pyocyaneus. 

Formation  of  Antitoxins. — It  was  formerly  believed  that  there  was  a 
direct  conversion  of  toxin  into  antitoxin,  but  this  certainly  is  not  the 
case,  for  the  amount  of  antitoxin  produced  is  altogether  out  of  propor- 
tion to  the  amount  of  toxin  injected. 

Antitoxins  are  formed  by  those  cells  that  anchor  the  toxins.  In 
order  to  produce  them  it  is  necessary  that  the  toxin  enter  into  direct 
union  with  the  cells  and  exert  a  stimulating  influence  on  them,  for  where 
a  loose  union  occurs,  as  between  cells  and  alkaloids,  antibodies  are  not 
formed. 

Having  entered  into  chemical  union  with  the  side-arms  of  cells, 
a  toxin  may  destroy  the  entire  cell,  and  if  a  sufficient  number  of 
these  are  destroyed,  the  host  will  show  symptoms  of  infection  and  may 
succumb.  If,  however,  the  cell  itself  is  not  destroyed,  but  only  one  or 
more  of  the  side-arms  injured,  the  damage  is  repaired  by  the  cell  form- 
ing new  side-arms  that  have  a  specific  affinity  for  the  toxin  responsible 
for  their  production.  According  to  Weigert's  overproduction  theory, 
a  cell  once  stimulated  to  produce  these  side-arms  or  receptors  continues 
to  produce  them  for  some  time,  even  after  the  stimulus  has  been  re- 
moved. In  this  manner  the  specific  receptors  are  produced  in  excess, 
and  since  all  cannot  remain  attached  to  the  parent  cell,  the  excess  is 
discharged  into  the  blood-stream.  Each  of  these  cast-off  receptors  is 
capable  of  uniting  with  toxin,  thus  neutralizing  the  poisonous  principles 

15 


22o 


ANTITOXINS 


the  toxin,  and  rendering  it  practically  harmless.  Antitoxins,  therefore, 
are  nothing  more  than  these  cast-off  receptors,  which  have  a  specific  affinity 
for  their  toxins  (Fig.  67). 

As  Adami  has  pointed  out,  it  is  probable  that  the  toxins  exist  for 
some  time  within  the  cell,  not  as  part  and  parcel  of  the  cell,  but  as  a 
stimulating  agent  that  causes  the  cell  to  develop  the  habit  of  producing 
the  specific  receptors.  The  mere  union  of  toxin  with  a  receptor,  causing 
it  to  fall  off,  and  being  followed  by  nature's  mode  of  repair,  with  the 
formation  of  an  excess  of  receptors  and  no  further  stimulation,  is  hardly 
sufficient  to  explain  the  enormous  activity  of  the  cells. 


FIG.  67. — THEORETIC  FORMATION  OF  ANTITOXINS. 

The  central  white  area  represents  a  molecule  of  a  cell;  the  shaded  portion  repre- 
sents the  cell  itself;  the  surrounding  area  represents  the  body-fluids  about  the  cell. 

r,  a  receptor  of  the  molecule  (first  order] ;  A,  overproduction  of  receptors,  which 
are  being  cast  oft7;  A2,  a  cast-off  receptor  free  in  the  body-fluids — now  an  antitoxin; 
A3,  a  molecule  of  antitoxin  combination  with  a  toxic  molecule  T3.  A3,  a  cast-off 
receptor  still  within  the  parent  cell;  T,  a  toxin  molecule  in  combination  with  the  re- 
ceptor of  a  cell  molecule;  T2,  a  toxin  molecule  free  in  the  body-fluids;  T3,  a  toxin 
molecule  in  combination  with  antitoxin;  T4,  a  molecule  of  toxoid  (toxophore  group 
lost). 


That  antitoxins  may  be  produced  locally  was  illustrated  by  the  ex- 
periment of  Romer  with  abrin.  This  substance  has  a  peculiarly  power- 
ful effect  upon  the  conjunctiva.  By  gradually  immunizing  the  right 
conjunctiva  of  a  rabbit  with  increasing  doses,  it  was  shown  that,  after 


PROPERTIES    OF   ANTITOXINS  227 

killing  the  animal  and  triturating  the  conjunctiva  with  a  fatal  dose  of 
abrin,  an  injection  of  the  emulsion  of  the  right  or  immunized  conjunctiva 
was  without  effect,  whereas  the  emulsion  from  the  left  proved  fatal. 
Thus  it  will  clearly  be  seen  that  the  cells  that  had  absorbed  the  abrin 
had  developed  and  contained  antiabrin  in  sufficient  amounts  to  neu- 
tralize the  poison. 

While  leukocytes,  such  as  Metchnikoff's  macrophages,  are  likewise 
active  in  the  formation  of  antitoxins,  it  is  certain  that  they  are  not  the 
only  cells  involved.  Metchnikoff  claims  that  antitoxins  are  merely 
toxins  altered  by  leukocytic  activity,  rather  than  constituents  of  tissue- 
cells;  this  explanation  is,  however,  inadequate,  and  it  has  been  shown 
experimentally  that  the  quantity  of  antitoxin  produced  is  so  far  in  ex- 
cess of  the  amount  of  toxin  injected  as  to  render  this  view  untenable. 

Structure  of  Antitoxins. — According  to  the  side-chain  theory,  anti- 
toxins are  the  simplest  of  antibodies,  being  composed  of  a  single  arm  or 
haptophore  group  for  union  with  the  toxin,  and  called  receptors  of  the 
first  order.  While  illustrations  of  this  theoretic  structure  will  convey 
the  impression  of  mere  physical  contact  or  union  with  toxin,  it  is  to  be 
remembered  that  experimental  data  indicate  that  the  union  and  con- 
sequent neutralization  of  the  toxin  are  chemical  .processes. 

Properties  of  Antitoxins. — While  chemical  analyses  to  determine 
the  nature  of  antitoxin  serums  were  made  as  early  as  1897,  little  is 
known  regarding  it  because  it  is  impossible  to  secure  the  antitoxic  ele- 
ment free  from  serum  and  serum  constituents.  Belfanti  and  Carbone 
found  that  most  of  the  antitoxin  in  a  serum  is  precipitated  with  the 
globulins  by  saturation  with  magnesium  sulphate.  This  work,  which 
has  been  verified  by  Atkinson  and  Pick,  shows  that  the  antitoxin  is 
carried  down  with  the  globulin  precipitates,  but  does  riot  necessarily 
prove  that  it  is  itself  a  globulin.  Later  Gibson  and  Banzhaf  showed  that 
the  portions  of  the  globulin  precipitate  soluble  in  saturated  sodium 
chlorid  solution  carried  most  of  the  antitoxin,  and  with  this  discovery 
a  practical  method  of  eliminating  much  of  the  non-antitoxic  portion  of 
the  serum  was  perfected. 

The  relation  of  antitoxins  to  proteids  has  also  been  studied,  digestive 
ferments  being  permitted  to  act  on  antitoxic  serum.  It  has  been  shown 
that  antitoxin  resists  tryptic  digestion  to  a  well-marked  degree;  in  this 
respect  it  resembles  the  serum  globulin.  All  the  evidence  obtained  in- 
dicates that  a  closer  relation  of  antitoxins  to  proteids  exists  than  has 
been  shown  for  the  toxins,  although  all  attempts  to  separate  antitoxins 
from  proteids  have  thus  far  failed. 


228  ANTITOXINS 

Antitoxins  are  fairly  resistant  bodies,  and  a  properly  prepared  anti- 
toxic serum,  when  kept  in  a  cool  place  and  protected  from  light  and  air, 
may  be  preserved  for  a  year  or  more  with  very  little  deterioration  in 
strength.  At  times,  however,  for  unknown  reasons,  antitoxins  gradu- 
ally deteriorate,  losing  about  2  per  cent,  in  strength  a  month.  Manu- 
facturers have  endeavored  to  calculate  this  loss  in  strength,  and  have 
placed  a  label  on  each  package  of  antitoxin,  bearing  a  date  beyond  which 
the  serum  is  not  guaranteed  to  contain  the  amount  of  antitoxin  present 
at  the  time  it  was  put  up. 

The  antitoxins,  with  few  exceptions,  are  far  more  stable  than  the 
toxins,  resisting -heating  up  to  62°  C.,  but  gradually  deteriorating  with 
higher  temperatures.  Boiling  destroys  them  completely.  They  are 
readily  preserved  with  small  amounts  of  chloroform,  phenol,  tricresol, 
etc.,  although  strong  solutions  of  these  produce  destructive  changes. 
Putrefaction  of  the  serum  destroys  the  antitoxin  content.  Ehrlich  has 
devised  the  best  method  for  their  preservation,  which  consists  in  drying  the 
serum  in  vacuo  and  preserving  it  in  the  dark,  at  a  low  temperature,  in  the 
presence  of  anhydrous  phosphoric  acid.  So  preserved,  antitoxin  retains 
its  strength  for  prolonged  periods  and  is  used  in  standardizing  toxins. 

Natural  Antitoxins. — The  appearance  of  so-called  natural  antitoxins 
can  be  explained  on  the  basis  of  Ehrlich's  theory.  Since  the  antitoxin 
is  composed  of  receptors  that  are  not  new  bodies,  but  simply  normal 
receptors  produced  in  excess,  it  is  reasonable  to  assume  that  a  few  may 
be  thrown  off  occasionally,  constituting  the  natural  antitoxin. 

Small  amounts  of  natural  diphtheria  antitoxin  may  be  found  in  cer- 
tain individuals  and  lower  animals.  Since  the  diphtheria  bacillus  is 
so  wide-spread  in  its  distribution,  it  is  possible  that  minor  subinfections 
may  be  responsible  for  antitoxin  production,  and  this  is  probably  always 
the  case  when  large  amounts  are  found. 

Information  regarding  natural  antitoxins  for  other  members  of  the 
toxin-producing  group  of  microorganisms  is  less  complete,  although  it 
is  highly  probable  that  natural  antitoxins  for  these  exist. 

The  Schick  Test  for  Natural  Diphtheria  Antitoxin. — Schick1  has 
worked  out  a  simple  and  practical  skin  test  which  apparent^  has  proved 
satisfactory  and  trustworthy  and  of  distinct  value  for  detecting  natural 
immunity  to  diphtheria  among  persons. 

This  test  consists  in  the  intradermic  injection  of  a  minute  dose  of 
diphtheria  toxin.  If  the  person  possesses  an  amount  of  antitoxin  equal 
to  at  least  one-thirtieth  of  a  unit  in  each  cubic  centimeter  of  blood-serum, 
1  Munch,  bed.  Woch.,  1913,  Ix,  2608. 


THE    SCHICK    TEST    FOR    NATURAL    DIPHTHERIA    ANTITOXIN 


229 


the  injected  toxin  is  neutralized  and  no  reaction  follows;  if,  however, 
the  person  does  not  have  antitoxin  in  the  body  fluids,  the  injected  toxin 
acts  as  an  irritant  to  the  skin,  producing  in  twenty-four  to  forty-eight 
hours  a  small  area  of  redness  and  edema.  A  positive  reaction  indicates 
that  the  person  does  not  possess  natural  antitoxin  in  his  blood,  and  there- 
fore that  he  is  susceptible  to  diphtheria;  a  negative  reaction  indicates 
that  natural  antitoxin  is  present  and  that  he  is,  in  all  probability,  immune 
to  diphtheria.  In  the  presence  of  exposure  to  diphtheria,  persons  reacting 
positively  to  .the  toxin  skin  test  should  receive  a  prophylactic  dose  of 
antitoxin,  while  those  reacting  negatively  may  with  safety  be  spared 
the  injection. 


FIG.  68. — METHOD  OF  ADMINISTERING  AN  INTRADERMAL  INJECTION  OP  DIPHTHERIA 

TOXIN  IN  THE  SCHICK  TEST. 

The  skin  has  been  cleansed  with  alcohol  and  pinched  up  between  the  thumb  and 
index-finger  of  the  left  hand;  the  needle  (No.  26)  has  been  entered  into  the  epidermis 
and  0.1  c.c.  of  fluid  injected.  Note  the  anemic  area  indicating  that  the  injection 
has  been  intradermic.  The  same  technic  is  employed  in  conducting  the  luetin  test.. 

It  will  be  understood,  therefore,  that  the  diphtheria  toxin  skin  reac- 
tion is  purely  an  inflammatory  reaction  due  to  the  fact  that  the  toxin  in  the 
skin  excites  inflammation  if  not  neutralized  with  diphtheria  antitoxin ;  it 
is  not  an  anaphylactic  reaction,  as  the  tuberculin  and  luetin  reactions. 

The  amount  of  toxin  to  be  injected  must  be  determined  beforehand 


230  ANTITOXINS 

in  the  laboratory.  A  series  of  guinea-pigs  weighing  from  250  to  300 
grams  are  injected  subcutaneously  with  increasing  doses  of  a  diphtheria 
toxin  to  determine  the  smallest  dose  that  will  just  kill  a  pig  at  the  end 
of  four  days  after  injection.  This  is  known  as  the  minimal  lethal  dose 
(M.L.D.),  and  one-fortieth  to  one-fiftieth  this  amount  is  the  proper  dose 
to  inject  into  the  skin.  To  facilitate  injecting  so  small  an  amount  of  fluid 
this  dose  is  so  diluted  with  sterile  normal  salt  solution  as  to  be  contained 
in  0.1  c.c.;  a  preservative,  as  0.25  per  cent,  tricresol  or  phenol,  is  added. 


FIG.  69. — SHOWING  THE  SMALL  ANEMIC  AREA  IMMEDIATELY  AFTER  AN  INTRACU- 

TANEOUS  INJECTION. 

The  toxin  does  not  contain  the  bacilli  and  this  dose  is  so  small  as  to 
be  without  danger. 

The  method  of  injection  is  very  important.  A  sterile  syringe  equipped 
with  a  fine  needle  is  required.  I  use  and  can  recommend  the  1  c.c.  Record 
syringe  fitted  with  No.  26  platinum  iridium  needle,  but  any  accurate 
syringe  so  made  that  0.1  c.c.  can  be  measured  and  injected  will  suffice. 
The  skin  of  the  upper  arm  is  wiped  off  with  a  pledget  of  cotton  and  al- 
cohol, the  skin  pinched  up  between  the  forefinger  and  thumb  of  the 
left  hand,  and  the  needle  entered  into  the  epidermis  and  not  under  it,  so 


FIG.  70. — THE  SCHICK  TEST  FOR  IMMUNITY  IN  DIPHTHERIA. 
Dr.  J.  A.  K.,  a  well-marked  reaction  thirty-six  hours  after  the  intracutaneous 
injection  of  one-fortieth  the  minimum  lethal  dose  of  a  diphtheria  toxin  diluted  to 
0.05  c.c.  The  patient's  blood  contained  no  antitoxin.  The  brownish  erythematous 
area  with  edematous  infiltration  of  the  subcutaneous  tissues  are  characteristic  of 
the  reaction.  (Reprinted  from  the  Amer.  Jour.  Dis.  Children.) 


THE    SCHICK   TEST    FOR    NATURAL   DIPHTHERIA    ANTITOXIN        231 

that  the  injection  is  intracutaneous  and  not  subcutaneous  (Fig.  68) .  When 
0.1  c.c.  is  injected  the  patient  should  experience  a  slight  stinging  sen- 
sation and  a  white  anemic  and  raised  spot,  resembling  a  wheal,  appears 
(Fig.  69).  If  this  spot  is  not  seen  the  injection  has  probably  been  subcu- 
taneous and  is  unsatisfactory. 

After  twenty-four  hours  a  positive  reaction  is  shown  by  an  area  of 
redness  and  induration.  The  experienced  observer  may  detect  a  reaction 
by  simply  running  the  finger  over  the  area  of  injection,  when  the  in- 
duration is  easily  felt.  Persons  reacting  negatively  show  nothing  more 
than  the  point  of  puncture,  and  at  times  a  very  small  area  of  redness 
measuring  |  inch  or  less  in  diameter  (Fig.  70). 

When  time  permits  it  is  better  practice  not  to  read  the  reaction  until 
forty-eight  hours  have  elapsed,  as  a  few  persons  will  show  a  false  reaction 
of  redness  and  slight  edema  at  the  end  of  twenty-four  hours  which  dis- 
appears after  forty-eight  hours,  whereas  the  true  toxin  reaction  remains 
or  may  slightly  enlarge  within  forty-eight  hours,  to  be  followed  by 
gradual  disappearance  of  all  signs  and  brownish  discoloration  and  slight 
.desquamation  of  the  epithelium,  lasting  several  weeks. 

In  a  special  study  with  Dr.  Moshage1  of  these  pseudoreactions  I 
have  found  that  they  are  to  be  largely  ascribed  to  a  peculiar  hypersensi- 
tiveness  of  the  skin  in  certain  individuals,  as  well  as  to  a  protein  reaction 
due  to  the  soluble  protein  constituents  of  bacilli  and  beef  in  the  toxin  as 
described  by  Park,  Serota,  and  Zingher.  For  this  reason  it  is  advisable 
to  use  highly  potent  toxins  requiring  high  dilution,  and  to  reduce  trauma 
as  much  as  possible  by  injecting  not  more  than  0.1  c.c.  as  the  dose. 

The  safety  and  practical  value  of  the  diphtheria  toxin  reaction  has 
been  established  since  announced  by  Schick  by  numerous  investigators 
in  this  country,  as  Park,  Zingher  and  Serota,2  Kolmer  and  Moshage,3 
Weaver  and  Maher,4  Graef  and  Ginsberg,5  Moody,6  Bundesen,  Moffett7 
and  Conrad,8  Linenthal  and  Rubin,9  as  well  as  by  several  European 
investigators. 

In  the  presence  of  an  outbreak  of  diphtheria,  therefor^,  the  physician 
should  take  cultures  and  apply  the  toxin  test  to  all  persons  exposed. 
At  the  end  of  twenty-four  hours  he  has  the  evidence  offered  by  both  at 

1  Jour.  Amer.  Med.  Assoc.,  1915,  65,  144.         2  Arch.  Pediat.,  1914,  xxxi,  481. 

3  Amer.  Jour.  Dis.,  Child.,  March,  1915,  189. 

4  Jour.  Infect.  Dis.,  1915,  xvi,  342. 

5  Jour.  Amer.  Med.  Assoc.,  1915,  64,  1205. 

6  Jour.  Amer.  Med.  Assoc.,  1915,  64,  1206. 

7  Jour.  Amer.  Med.  Assoc.,  1915,  64,  1203. 

8  Jour.  Amer.  Med.  Assoc.,  1915,  65,  1010. 

9  Boston  Med.  and  Surg.  Jour.,  1915,  clxxiii,  No.  12. 


232  ANTITOXINS 

hand.  This  delay  of  twenty-four  hours  before  immunizing  with  anti- 
toxin is  justifiable  unless  a  contact  shows  clinical  signs  suggestive  of 
diphtheria.  In  such  an  instance  we  believe  that  antitoxin  should  be 
given  as  soon  as  possible. 

The  test  has  a  special  field  of  usefulness  in  hospitals  and  institutions 
for  the  care  of  children.  All  children  should  be  subjected  to  the  toxin  test 
prior  to  admission,  and  those  immunized  who  react  positively. 

I  have  applied  this  test  to  over  700  persons  of  various  ages  with  the 
following  reactions: 

Under  1  year 15  per  cent,  positive;        85  per  cent,  negative. 

Ito    2  years 43  "  "  57 

2  to    4  years. 66  "  "  34 

4  to    6  years 58  "  "  42 

6  to    8  years 51  "  "  49 

8  to  15  years 24  "  "  76 

15  to  30  years 40  "  "  60        " 

Over  30  years 26  "  "  74 

In  scarlet  fever,  measles,  and  acute  anterior  poliomyelitis  the  per- 
centage of  positive  reactions  is  increased. 

The  percentage  of  negative  reactions  is  of  particular  interest  as  indi- 
cating the  number  of  persons  who  are  naturally  immune  to  diphtheria 
on  the  evidence  of  this  test,  and  showing  the  value  of  the  reaction,  es- 
pecially in  institutions  for  children  where  a  large  number  may  be  exposed, 
and  requiring  prophylactic  injection  of  antitoxin  unless  known  to  be 
naturally  immune  to  the  disease.  If  it  is  the  custom  to  reinject  with 
antitoxin  a  month  or  six  weeks  later,  it  is  better  practice  to  apply  the 
toxin  test  first  and  administer  serum  to  those  only  who  show  a  positive 
reaction.  This  means  a  saving  of  antitoxin  and  the  avoidance  of  the 
discomfort  attending  the  injection  of  serum  and  disagreeable  serum 
sickness. 

Specificity  of  Antitoxins. — Antitoxins  well  illustrate  the  law  of 
specificity  that  exists  between  antigen  and  antibody,  since  they  are 
strictly  specific  for  their  toxins.  Diphtheria  antitoxin  will  neutralize 
only  diphtheria  toxin;  tetanus  antitoxin,  only  tetanus  toxin,  and  so  on 
through  the  list.  This  specificity  is  not  confined  to  the  particular 
toxin-producing  organism  that  generates  the  antitoxin;  for  example, 
there  are  various  kinds  of  diphtheria  bacilli,  differing  as  regards 
morphology  and  toxicity,  although  one  antitoxin  appears  to  act  the  same 
with  their  various  toxins. 

Nature  of  the  Toxin- Antitoxin  Reaction. — While  the  injection  of  toxin- 
antitoxin  mixtures  into  the  lower  animals  is  the  only  practical  method 
of  testing  and  standardizing  the  curative  and  prophylactic  powers  of 


NATURE    OF   THE    TOXIN-ANTITOXIN    REACTION  233 

their  serums,  this  method  does  not  throw  much  light  upon  the  nature  of 
the  toxin-antitoxin  reaction,  or  show  how  antitoxin  overcomes  the  toxin. 

Antitoxin  is  protective  and  curative,  in  that  it  actually  destroys  the 
toxin,  in  a  manner  similar  to  the  dissolution  of  a  bacterium  caused  by  a 
specific  bacteriolysin:  or  it  may  influence  the  tissue-cells  in  some  way  and 
render  them  more  resistant  to  the  toxins,  a  view  that  was  held  by 
Roux,  and  particularly  by  Buchner;  or  the  antitoxin  may  form  a  di- 
rect chemical  union  with  the  toxin,  similar  to  the  chemical  neutraliza- 
tion of  an  acid  by  a  base — an  opinion  early  held  by  Behring  and  elabora- 
ted later  by  Ehrlich. 

Experimental  data  support  the  view  of  chemical  union  with  the 
toxin.  In  the  test-tube  some  time  is  required  for  the  union  of  toxin  and 
antitoxin  to  occur;  this  union  is  hastened  by  heat  and  retarded  by  cold; 
it  is  more  rapid  in  concentrated  than  in  dilute  solutions,  and  in  general 
takes  place  in  accordance  with  the  law  of  multiple  proportions — all 
of  which  tends  to  show  the  close  similarity  of  the  fcoxin-antitoxin  reaction 
to  a  chemical  process. 

It  is  generally  conceded  that  antitoxin  does  not  directly  destroy  the 
toxin,  for  when  neutral  mixtures  of  toxin  and  antitoxin  are  injected  into 
animals,  portions  of  toxin  may  become  dissociated  and  unite  with  tissue- 
cells  possessing  greater  affinity  for  the  toxin,  and  symptoms  of  infection 
may  result.  It  is  probable  that  toxin  and  antitoxin  form  a  distinct 
compound,  and  this  action  requires  time  for  its  consummation.  For 
example,  Martin  and  Cherry,  by  filtering  mixtures  of  toxin  and  anti- 
toxin through  fine  filters  that  would  permit  the  toxin  molecule  to  pass 
through  but  restrain  the  larger  antitoxin  molecule,  found  that,  if  filtered 
immediately,  all  the  toxin  in  the  mixtures  was  extruded,  but  that,  as  the 
interval  between  mixing  and  filtration  was  prolonged,  less  and  less  toxin 
appeared  in  the  filtrate,  until  finally,  two  hours  after  mixing,  no  toxin 
whatever  passed  through  the  filter. 

This  element  of  time  in  support  of  the  chemical  nature  of  the  reaction 
is  further  strengthened  by  the  experiments  of  Calmette  with  snake  venom 
and  antivenin,  and  likewise  serves  to  demonstrate  that  the  antitoxin 
apparently  does  not  directly  destroy  the  toxin.  Although  most  toxins 
are  thermolabile,  Calmette  found  that  snake  venom  is  rendered  inert 
by  heating  to  68°  C.,  whereas  the  antivenin  remains  uninfluenced  by  a 
temperature  of  80°  C.  When  neutral  mixtures  of  venom-antivenin  were 
heated  to  70°  C.,  they  were  found  to  become  toxic  again,  presumably 
on  account  of  the  destruction  of  the  antivenin,  the  venom  itself  not 
being  destroyed.  Similar  experiments  were  carried  out  by  Wassermann 
with  mixtures  of  pyocyaneus  toxin-antitoxin,  with  similar  results.  In 


234  ANTITOXINS 

both  instances,  however,  as  developed  later,  if  the  mixtures  had  been 
allowed  to  stand  longer,  these  results  would  not  have  been  secured.  Al- 
though performed  originally  to  sho*w  that  an  antitoxin  does  not  act  by 
actually  destroying  its  toxin,  these  experiments  simply  demonstrate  the 
importance  of  the  element  of  time  in  the  reaction,  without  throwing  any 
real  light  upon  the  nature  of  the  new  toxin-antitoxin  compound,  if  such 
exists. 

That  toxin  is  counteracted  by  antitoxin,  independent  of  the  partici- 
pation of  living  tissue-cells,  has  been  quite  conclusively  proved  by  ex- 
periments in  vitro.  Ehrlich  showed  that  the  agglutinating  qualities  of 
ricin — a  vegetable  toxin — may  be  overcome  in  the  test-tube  by  adding 
antiricin,  the  corresponding  antitoxin.  Similar  results  were  obtained 
by  Ehrlich  with  tetanolysin  and  tetanus  antitoxin,  and  by  Stephens 
and  Myers  with  cobra  venom  and  its  antivenin. 

It  is  probable  that  antitoxin  has  a  similar  action  when  injected  for 
therapeutic  purposes,  as  for  curing  an  infection.  The  longer  the  in- 
terval that  has  elapsed  between  the  time  of  infection  and  the  administra- 
tion of  antitoxin,  the  less  satisfactory  will  be  the  result,  as  antitoxin 
becomes  less  powerful  when  toxins  have  formed  a  firm  union  with  the 
body-cells.  This  is  especially  true  in  tetanus,  where  even  very  large 
doses  of  antitoxin  may  be  incapable  of  dissociating  the  toxin  molecule 
from  the  nerve-cells,  the  serum,  therefore,  being  of  greatest  value  in 
prophylaxis.  In  diphtheria,  however,  the  union  between  toxin  and  cells 
is  less  firm,  and  the  antitoxin  is  probably  capable  of  neutralizing  the 
toxin  already  present  in  the  cells,  and  especially  any  toxin  that  may 
become  dissociated  from  the  cell  or  is  freshly  prepared  by  the  diphtheria 
bacillus  at  the  site  of  infection.  The  indication,  therefore,  in  giving 
antitoxin,  is  to  give  a  dose  large  enough  to  neutralize  all  free  and  loosely 
bound  toxin,  with  an  excess  to  neutralize  dissociated  toxin  and  that  pre- 
pared by  the  bacillus  during  the  course  of  the  infection. 

The  introduction  of  the  test-tube  experiment  into  the  investigation 
of  these  reactions  permitted  more  exact  observations  to  be  made,  and 
the  evidence  secured  by  this  means,  as  well  as  by  carefully  graded  quan- 
titative animal  experiments,  would  seem  to  indicate  that  we  should  ac- 
cept, for  the  present  at  least,  the  conception  of  the  chemical  nature  of 
the  process. 


PRODUCTION  OF  ANTITOXINS  FOR  THERAPEUTIC  PURPOSES 

Diphtheria  and  tetanus  antitoxins  are  manufactured  on   a   large 
scale,  and  are  used  extensively  in  the  prevention  and  cure  of  these  in- 


PRODUCTION    OF    ANTITOXINS    FOR    THERAPEUTIC    PURPOSES    235 

fections.  They  are  prepared  by  immunizing  horses  with  carefully 
graded  and  increasing  doses  of  the  respective  toxins  until  the  serum  of 
the  animals  shows  a  sufficiently  high  antitoxin  content,  after  prelim- 
inary trials,  to  warrant  more  extensive  bleeding.  Large  quantities  of 
blood  are  then  collected  aseptically  by  puncturing  the  jugular  vein. 
The  serum  is  carefully  separated  and  standardized  according  to  an  ac- 
cepted technic,  in  order  to  determine  the  antitoxin  content  in  units. 
A  small  amount  of  preservative  is  added,  and  the  serum  is  finally  dis- 
pensed in  special  containers  or  syringes  ready  for  administration.  In 
some  laboratories  it  is  customary  to  precipitate  the  globulin  fraction  of 
the  serum  with  magnesium  or  ammonium  sulphate,  and  redissolve  the 
portion  containing  most  of  the  antitoxin  in  saturated  sodium  chlorid 
solution.  The  bulk  of  the  serum  is  thus  greatly  decreased,  and  ob- 
jectionable constituents  largely  eliminated,  to  the  obvious  advantage 
of  the  preparation  for  therapeutic  purposes. 

Antitoxins  have  also  been  prepared  for  other  bacterial  toxins,  as 
those  of  the  dysentery  bacillus  (Kruse-Shiga)  and  Bacillus  botulinus, 
for  the  vegetable  toxins  in  pollen,  and  for  the  animal  toxins  in  snake  ven- 
oms. 

There  are  other  serums  for  the  treatment  of  certain  infections,  which 
depend  for  their  effects  chiefly  upon  the  presence  of  bacteriolysins  and 
immune  opsonins,  and  these  are  described  in  a  subsequent  chapter. 

THE  PRODUCTION  OF  DIPHTHERIA  ANTITOXIN 

The  following,  taken  largely  from  Park,  is  a  widely  used  and  ac- 
cepted technic  for  the  production  of  diphtheria  antitoxin: 

Production  of  the  Diphtheria  Toxin. — A  strong  diphtheria  toxin 
should  be  obtained  by  growing  a  virulent  culture  in  a  2  per  cent,  nutri- 
ent peptone  bouillon  made  from  "bob"  veal,  of  an  alkalinity  that  should 
be  about  9  c.c.  of  normal  soda  solution  per  liter  above  the  neutral  point 
to  litmus,  and  prepared  from  a  suitable  peptone  (Witte).  The  broth 
should  be  poured  into  large-necked  Erlenmeyer  flasks  in  comparatively 
shallow  layers,  so  as  to  allow  of  the  free  access  of  air,  and  maintained  at  a 
temperature  of  about  35°  to  36°  C.  (Fig.  71). 

In  the  Hygienic  Laboratory  of  the  Public  Health  and  Marine-Hos- 
pital Service  "Smith's  bouillon"  is  used  for  preparing  the  toxin.  This 
is  made  of  fresh  lean  beef,  after  the  muscle  sugar  and  all  other  sugars 
have  been  removed  by  fermentation  with  a  good  culture  of  Bacillus  coli. 
The  reaction  is  adjusted  until  0.5  per  cent,  acid  to  phenolphthalein,  that 
is  still  distinctly  alkaline  to  litmus,  and  1  per  cent,  peptone,  0.5  per  cent. 


236  ANTITOXINS 

sodium  chlorid,  and  0.1  per  cent,  dextrose  are  added.  The  reaction  is 
again  noted,  and  adjusted  to  +  0.5  per  cent.  The  broth  is  then  filtered 
through  filter-paper  into  flasks  and  test-tubes  and  sterilized  in  the  auto- 
clave at  a  temperature  of  120°  C.  for  twenty  minutes. 

After  incubating  for  from  seven  to  ten  days  the  culture  is  removed, 
and  its  purity  having  been  tested  by  microscopic  and  cultural  methods, 
it  is  rendered  sterile  by  the  addition  of  10  per  cent,  of  a  5  per  cent,  solu- 
tion of  carbolic  acid.  After  forty-eight  hours  the  dead  bacilli  have 
settled  on  the  bottom  of  the  jar,  and  the  clear  fluid  above  is  siphoned 
off,  filtered,  and  stored  in  full  bottles  in  a  cold  place  until  needed  (Fig. 
72). 


FIG.  71. — A  FLASK  OF  DIPHTHERIA  CULTURE. 

The  bacilli  grow  on  the  surface  and  form  a  scum.  As  the  culture  grows  older, 
the  bacilli  die  and  sink  to  the  bottom  of  the  flask.  A  flask  of  this  shape  affords  a 
large  surface  of  culture-medium  in  contact  with  oxygen  and  facilitates  toxin  pro- 
duction. 

Testing  the  Toxin. — The  strength  of  the  toxin  is  then  tested  by 
injecting  a  series  of  guinea-pigs  with  carefully  measured  amounts. 
When  injected  hypodermically,  less  than  0.005  c.c.  should  kill  a  250- 
gram  guinea-pig,  and  a  toxin  requiring  more  than  0.01  c.c.  to  kill  a  pig 
of  this  weight  is  too  weak  for  present  purposes.  This  preliminary  titra- 
tion  of  the  toxin  will  suffice  for  determining  the  dosage  for  horses,  but 
in  standardizing  antitoxin,  the  technic  must  necessarily  be  more  ac- 
curate. 

Immunizing  the  Animals. — The  horses  used  should  be  young,  vig- 
orous, of  fair  size,  and  absolutely  healthy.  They  should  be  severally 


PRODUCTION  OF  ANTITOXINS  FOR  THERAPEUTIC  PURPOSES   237 

injected  with  10,000  units  of  antitoxin  and  the  following  day  with  5000 
units  of  toxin,  an  amount  sufficient  to  kill  5000  guinea-pigs  each  weighing 


FIG.  72. — A  LARGE  TOXIN  FILTER. 

The  culture  is  contained  in  the  large  bottle  on  the  shelf,  and  drains  into  the  flask, 
which  in  turn  empties  into  the  earthen  "candle."  By  means  of  a  vacuum  the  culture 
is  filtered  through  the  "  candle"  and  collects  in  the  large  bottle  at  the  base  of  the  stand. 

250  grams.    If  antitoxin  is  not  given  with  the  first  doses  of  toxin,  only  one- 
tenth  of  the  dose  advised  is  to  be  given.    After  from  three  to  four  days,  or  as 


238  ANTITOXINS 

soon  as  the  temperature  reaction  has  subsided,  a  second  subcutaneous 
injection  of  a  slightly  larger  dose  is  given,  the  amount  of  toxin  increasing 
about  10  to  15  c.c.  per  dose  until,  six  weeks  later,  the  animal  is  receiving 
from  20  to  30  times,  and  on  the  sixtieth  day  about  60  times,  the  amount 
originally  given.  At  the- end  of  this  time  a  trial  bleeding  is  made  and 
the  serum  tested. 

There  is  absolutely  no  way  of  judging  which  horses  will  produce  the 
highest  grades  of  antitoxin.  Roughly  estimated,  those  horses  that  are 
extremely  sensitive  and  those  that  react  feebly  are  the  poorest,  but 
there  are  exceptions  even  in  these  cases.  The  only  reliable  method, 
therefore,  is  to  bleed  the  horses  at  the  end  of  six  weeks  or  two  months 
and  test  their  serum.  As  shown  by  Park  and  Zingher,  persons  yielding 
negative  Schick  tests  respond  to  toxin-antitoxin  injections  with  the 
production  of  antitoxin  more  readily  than  persons  who  react  in  a  positive 
manner.  Taking  advantage  of  this  data,  Hitchens  and  Tingley  have 
injected  0.2  c.c.  diphtheria  toxin  equal  to  3  M.L.D.  for  250-gram  pigs 
into  the  conjunctiva  of  one  eye  of  horses  under  examination  for  the 
purposes  of  immunization,  and  found  that  many  yielded  negative  tests 
read  at  the  end  of  forty-eight  hours,  indicating  the  presence  of  natural 
diphtheria  antitoxin  in  the  blood  of  normal  horses;  these  animals  are 
probably  to  be  preferred  in  the  production  of  antitoxin,  as  they  are 
likely  to  yield  highly  potent  sera.  If  only  high-grade  serum  is  wanted, 
all  horses  that  give  less  than  150  units  per  cubic  centimeter  should  be 
discarded.  The  remaining  horses  should  receive  steadily  increasing 
doses,  the  rapidity  of  the  increase  and  the  interval  of  time  between  the 
doses  (three  days  to  one  week)  depending  somewhat  on  the  reaction 
following  the  injection,  an  elevation  of  temperature  of  more  than  3°  F. 
being  undesirable. 

For  example,  according  to  Park,  a  horse  that  yielded  an  unusually 
high  grade  of  serum  was  started  on  12  c.c.  of  toxin  (xuir  c.c.  fatal  dose), 
together  with  10,000  units  of  antitoxin.  Sixty  days  later  a  dose  of  675 
c.c.  was  given,  and  the  serum  contained  1000  units  of  antitoxin  per 
cubic  centimeter.  Regular  bleedings  were  made  weekly  for  the  next 
four  months,  at  the  end  of  which  time  the  serum  had  fallen  to  500  units 
in  spite  of  weekly  gradually  increasing  doses  of  toxin.  At  the  end  of 
three  months  the  antitoxic  serum  of  all  the  horses  should  contain  over 
300  units,  and  in  about  10  per  cent,  as  much  as  800  units  in  each  cubic 
centimeter.  Not  more  than  1  per  cent,  give  above  1000  units,  and, 
according  to  Park,  so  far  none  has  given  him  as  much  as  2000  units  per 
cubic  centimeter.  The  very  best  horses,  if  pushed  to  their  limit,  con- 


FIG.    73. — PREPARATION    OF    DIPHTHERIA    ANTITOXIN.     SEPARATION    OF    BLOOD- 
SERUM. 

The  bottle  on  the  left  shows  blood  after  standing  about  an  hour;  the  bottle  on  the 
right  shows  the  separation  of  serum  about  twelve  hours  after  bleeding. 


PRODUCTION    OF   ANTITOXINS    FOR   THERAPEUTIC    PURPOSES       239 

tinue  to  furnish  blood  containing  the  maximum  amount  of  antitoxin 
for  several  months,  and  then,  in  spite  of  increasing  injections  of  toxin, 
begin  to  furnish  blood  of  gradually  decreasing  strength.  If  an  interval 
of  three  months'  freedom  from  inoculation  is  allowed  once  every  nine 
months,  the  best  horses  will  furnish  high-grade  serum  for  from  two  to 
four  years. 

Collecting  the  Serum. — In  order  to  obtain  the  serum,  the  neck  of 
the  horse  should  be  cleansed  thoroughly  as  for  an  aseptic  operation,  and 
a  special  tourniquet  applied  to  distend  the  jugular  vein.  A  small  slit 
is  made  through  the  skin  over  the  vein,  and  a  special  sharp-pointed 
cannula  is  passed  upward  under  the  skin  for  two  inches  or  more  and 
then  plunged  into  the  vein.  From  6  to  12  liters  of  blood  are  collected 
by  a  rubber  tube  into  cylindric  jars  provided  with  special  tops,  facilitat- 
ing filling  with  blood  and  subsequent  withdrawal  of  the  serum.  The 
cannula,  tubing,  jars,  and  everything  used  in  collecting  the  blood  and 
serum  should  be  carefully  sterilized,  and  the  whole  operation  should 
be  conducted  with  scrupulous  aseptic  care  in  order  to  avoid  contamina- 
tion. (See  Fig.  26.) 

The  jars  are  set  aside  (Fig.  73)  for  three  or  four  days,  and  the  serum 
is  drawn  off  by  means  of  sterile  glass  and  rubber  tubing  and  stored  in 
large  sterile  bottles.  When  the  globulins  are  to  be  separated,  the  blood 
may  be  added  directly  to  one-tenth  of  its  volume  of  a  10  per  cent,  solu- 
tion of  sodium  citrate,  which  prevents  clotting  of  the  blood. 

The  serum  should  be  clear  and  free  from  blood,  and  its  sterility 
should  be  proved  by  culture  tests.  An  antiseptic,  such  as  0.4  per  cent, 
tricresol,  0.5  per  cent,  phenol,  or  chloroform,  may  be  added,  but  this  is 
not  necessary  unless  it  is  desired  to  keep  the  serum  for  some  time.  The 
serum  is  poured  into  small  bottles  fitted  with  rubber  stoppers,  or  placed 
in  special  syringes  labeled  with  the  number  of  units  contained.  The 
whole-  process  should  be  conducted  with  scrupulous  aseptic  technic. 
Diphtheria  toxin  varies  too  much  to  be  used  as  a  standard  in  determin- 
ing the  antitoxin  content  of  a  serum;  hence  a  dried  antitoxin  is  pre- 
pared by  the  Hygienic  Laboratory  and  is  distributed  for  this  purpose. 
The  serum  is  evaporated  and  dried  in  vacuo  by  passing  dry  sterile  air 
heated  to  35°  C.  through  it,  and  when  perfectly  dry,  is  preserved  in 
special  containers  over  anhydrous  phosphoric  acid  at  a  constant  temper- 
ature of  5°  C.  Preserved  in  this  manner,  the  antitoxin  is  quite  stable. 
Just  before  use  it  is  dissolved  in  the  required  amount  of  sterile  normal 
salt  solution. 

Method  of  Concentrating  Serum  by  Isolating  the  Antitoxin  Globu- 


240  ANTITOXINS 

lins. — The  use  of  concentrated  serum  has  lessened  the  incidence  of  serum 
sickness  and  facilitates  the  administration  of  large  doses.  Briefly /'the 
method  of  Banzhaf  is  as  follows:  "The  citrated  plasma  is  diluted  with 
half  its  volume  of  water  and  saturated  ammonium  sulphate  solution  is 
added  up  to  30  per  cent,  saturated  solution.  This  mixture  is  heated  up 
to  60°  C.  and  held  there  for  one  hour.  Then  filtered  while  hot.  The 
precipate  contains  the  native  non-antitoxic  proteins  and  a  large  amount 
of  non-antitoxic  proteins  newly  formed  by  the  above  method  of  heating. 
This  precipitate  is  discarded.  The  nitrate  is  brought  up  to  50  per  cent, 
saturated  ammonium  sulphate  solution.  The  resulting  precipitate 
contains  only  pseudoglobulin  and  antitoxin  and  is  pressed  to  remove 
excess  of  fluid,  followed  by  dialyzation  until  free  from  salts.  After 
dialysis  is  completed  0.8  per  cent,  sodium  chlorid  is  added  for  isotox- 
icity  and  0.3  per  cent,  tricresol  for  preservation.  It  is  then  filtered 
through  paper  pulp  and  a  Berkefeld  clay  filter,  tested  for  sterility  and 
potency,  and  filled  into  sterile  syringes  or  bottles.  This  method  gives  a 
concentration  of  about  six  times  the  original  potency"  (Park). 

Standardizing  the  Serum. — During  the  earlier  investigations  it  was 
believed  that  toxin  was  quite  stable,  and  that  it  possessed  a  definite 
toxicity  with  a  constant  value  in  neutralizing  antitoxin.  Upon  these 
suppositions  the  original  Behring-Ehrlich  antitoxin  unit  was  based, 
consisting  of  10  times  the  amount  of  antitoxin  that  neutralized  10  fatal 
doses  of  toxin.  For  example,  if  the  minimal  lethal  dose  (M.  L.  D.)  of 
toxin  was  0.001  c.c.,  and  0.01  c.c.  was  neutralized  by  0.01  c.c.  of  serum, 
then  0.1  c.c.  of  serum  equaled' one  unit,  or  10  units  in  a  cubic  centimeter. 
Later  stronger  serums  were  found,  and  von  Behring  and  Ehrlich  modi- 
fied the  unit,  which  they  now  call  the  immunity  unit,  to  be  that  quantity 
of  antitoxin  which  will  neutralize  100  times  the  minimal  fatal  dose  for  a 
250-gram  guinea-pig. 

It  was  soon  discovered  that  toxins  are  unstable  compounds,  and  that, 
almost  immediately  after  their  production,  they  begin  to  change  into 
toxoids,  which  are  not  acutely  poisonous,  but  which  retain  their  power 
to  neutralize  antitoxin. 

In  order  to  standardize  a  serum  it  is  necessary  that  the  strength  of 
the  toxin  be  known,  and  since  this  is  so  variable,  a  standard  antitoxin  is 
supplied  by  the  Hygienic  Laboratory,  by  which  the  various  antitoxin 
plants  may  measure  the  strength  of  their  toxins.  By  mixing  varying 
quantities  of  toxin  with  one  unit  of  this  standard  antitoxin  and  injecting 
these  into  250-gram  guinea-pigs,  the  L+  (limes  death)  dose  is  obtained, 
which  is  the  dose  of  toxin  required  to  kill  a  pig  in  four  days  with  the  one 


PRODUCTION  OF  ANTITOXINS  FOR  THERAPEUTIC  PURPOSES   241 

unit  of  antitoxin.  In  order  accurately  to  determine  this  dose  many 
pigs  may  be  required,  but  this  method  of  titration  is  the  key-note  to 
successful  standardization. 

Such  a  titration  for  instance,  has  shown  a  toxin  to  react  as  follows : 


TABLE   1.— METHOD    OF   DETERMINING   THE    L+   DOSE    OF   DIPH- 
THERIA TOXIN 


One  antitoxin  unit+0.2 
+0.22 
+0.24 
+0.25 
+0.26 
+0.28 
+0.3 
+0.32 
+0.34 
+0.35 


c.c.  toxin  —  No  visible  symptoms. 
=  No  symptoms. 

=  Usually  no  symptoms  or  a  very  slight  reaction. 
=  Very  slight  congestion  and  edema. 
=  Slight  edema  at  site. 
=  Edema;  sometimes  late  paralysis. 
=  Acute  edema  and  sometimes  death. 
=  Always  acute  death  about  the  fourth  day. 
=  Death  from  second  to  third  day. 
'        '     =  Death  about  the  second  day. 


Here  the  L+  dose  is  0.32  c.c.  The  dose  of  toxin  that  just  neu- 
tralizes the  antitoxin  without  causing  symptoms  has  been  called  by 
Ehrlich  the  L6  (limes  zero)  dose,  and  in  this  instance  it  is  about  0.24  c.c. 
This  determination,  however,  has  not  the  same  practical  value  as  the 
Ii  dose. 


FIG.  74.— A  KITCHENS  SYRINGE. 

The  needle  is  plugged  by  dipping  the  tip  in  carbolized  vaselin.  The  side  arm 
holds  sterile  salt  solution;  when  the  needle  has  been  entered,  the  injection  is  given  by 
pressure  on  the  bulb;  the  side  arm  is  then  turned  upward,  when  the  contents  flow 
into  the  main  barrel,  and  injected  in  this  manner  insures  accuracy  in  dosage  and 
uniform  bulk  of  inoculum. 

Having  determined  the  L-j.  dose  of  the  toxin,  a  series  of  six  to  eight 
guinea-pigs  are  injected  with  this  constant  dose  of  toxin  and  increasing 
amounts  of  the  corresponding  antitoxin  serum;  for  instance,  No.  1 
would  receive  0.001  c.c.  of  serum;  No.  2,  0.002  c.c.;  No.  3,  0.003  c.c.; 
No.  4,  0.004  c.c.;  No.  5,  0.005  c.c.;  No.  6,  0.006  c.c.,  etc.  If  at  the  end 
16 


242 


ANTITOXINS 


of  the  fourth  day  Nos.  1,  2,  3,  and  4  were  dead  and  Nos.  5  and  6  were 
alive,  the  serum  would  contain  200  units  of  antitoxin  in  a  cubic  centi- 
meter. These  injections  are  best  given  with  precision  syringes,  the  one 
devised  by  Hitchens  being  particularly  serviceable  (Fig.  74).  The  syr- 
inges are  sterilized,  and  the  needles  are  dipped  in  sterile  vaselin  to 
plug  them.  The  mixtures  are  made  in  the  barrel  of  the  syringe,  and 
sufficient  sterile  salt  solution  is  placed  in  the  side-arm  to  bring  the  total 
volume  of  the  injection  up  to  4  c.c.,  and  to  wash  in  all  traces  of  toxin  and 
antitoxin.  The  mixtures  are  allowed  to  stand  for  at  least  fifteen  min- 
utes (Park)  before  being  injected  (Fig.  75).  The  pigs  must  be  of  proper 
weight — i.  e.,  about  250  to  300  grams;  the  abdominal  wall  is  shaved, 


FIG.  75. — A  BATTERY  OF  HITCHENS  SYRINGES. 

and  the  injection  given  directly  in  the  median  abdominal  line.  The 
animals  are  placed  two  in  a  cage,  and  carefully  observed  for  four  or 
five  days  for  symptoms  of  toxemia  and  edema  about  the  site  of  injection. 

Romer's  Method  of  Determining  Small  Amounts  of  Diphtheria  Antitoxin. — The 

principle  of  this  method  is  based  upon  the  observation  that,  when  very  small  amounts 
of  diphtheria  toxin  are  injected  intracutaneously  into  the  abdominal  skin  of  guinea- 
pigs,  small  areas  of  edema  and  necrosis  result  in  about  forty-eight  hours.  When 
such  injections  are  made  with  mixtures  of  toxin  and  antitoxin,  the  presence  of  free 
toxin  is  indicated  by  such  tissue  changes.  It  is  chiefly  used  in  determining  the  anti- 
toxin content  of  human  serums  after  active  immunization  with  the  toxin-antitoxin 
mixtures  of  von  Behring.  (See  p.  770.) 

Technid. — I  conduct  this  test  in  the  following  manner:  The  "limes-necrosis" 
(Ln)  dose  of  a  toxin  is  first  determined,  which  is  the  amount  of  toxin  which,  together 
with  TFJHT  of  a  unit  of  standard  antitoxin,  will  still  produce  a  minimal  amount  of  ne- 
crosis in  forty-eight  hours  after  intracutaneous  injection  into  .guinea-pigs.  A  series  of 
dilutions  of  the  L+  dose  of  a  toxin  is  made,  ranging  from  1  :  5  to  1  :  100,  and  0.2 
c.c.  of  each  mixed  with  0.2  c.c.  of  antitoxin  so  diluted  that  each  0.1  c.c.  contains 


PRODUCTION    OF   TETANUS   ANTITOXIN  243 

i  ATT  of  a  unit.  These  mixtures  are  made  in  small  test-tubes,  the  cotton  stoppers 
paraffined,  and  the  tubes  incubated  for  three  hours  and  placed  in  the  refrigerator  for 
twenty-one  hours,  after  which  0.2  c.c.  of  each  is  injected  into  guinea-pigs  (prepared 
by  pulling  out  the  hairs);  several  injections  may  be  made  in  each  pig. 

When  the  Ln  dose  of  the  toxin  has  been  determined  this  amount  is  mixed  in  a 
similar  manner  with  varying  amounts  of  the  patient's  serum  being  tested.  The 
amount  of  serum  just  neutralizing  the  toxin  contains  roVs"  °f  a  unit  of  antitoxin  from 
which  the  amount  of  antitoxin  per  cubic  centimeter  of  serum  may  be  computed.  For 
example  I  have  found  that  0.003  c.c.  of  serum  of  a  person  reacting  negatively  to  the 
Schick  test  neutralized  this  amount  of  toxin;  therefore  each  cubic  centimeter  of 
this  person's  serum  contained  0.33  unit  of  antitoxin. 


PRODUCTION  OF  TETANUS  ANTITOXIN 

The  method  used  in  the  production  of  tetanus  antitoxin  is  similar  to 
that  employed  in  producing  diphtheria-antitoxin,  the  horses  being 
inoculated  with  increasing  doses  of  a  strong  tetanus  toxin. 

Tetanus  Toxin. — The  toxin  is  secured  by  inoculating  large  flasks 
or  tubes  of  neutral  veal  broth  containing  1  per  cent,  of  sodium  chlorid 
and  peptone  with  abundant  tetanus  culture,  and  growing  these  anae- 
robically  at  37°  C.  for  two  weeks.  Anderson  and  Leake1  prepare  100 
liters  of  broth  with  50  kilograms  of  minced  round  steak  and  add  0.5 
per  cent,  sodium  chlorid  and  1  per  cent,  peptone;  after  steaming  for  an 
hour  the  reaction  is  made  neutral  to  phenolphthalein  and  the  broth 
filtered  through  paper  into  liter  Erlenmeyer  flasks,  followed  by  steaming 
without  pressure  for  one  and  one-half  hours.  The  broth  may  be  stored 
for  a  period  of  two  weeks  or  less.  Just  before  inoculating,  a  1  per  cent, 
solution  of  C.  P.  glucose  (powdered)  is  added  and  the  medium  again 
heated  for  one  and  one-half  hours  without  pressure,  cooled  to  40°  C., 
and  immediately  inoculated  a  few  centimeters  below  the  surface  with 
1  c.c.  of  a  twenty-four-hour  broth  culture  of  tetanus  bacilli  which  has 
been  subcultured  daily  in  1  per  cent,  glucose  broth  for  one  to  three  weeks. 
No  oil  or  other  means  is  used  to  secure  anaerobiasis;  incubation  is  al- 
lowed to  go  on  undisturbed  for  fifteen  days  at  37°  C.  The  cultures  are 
then  filtered  rapidly  through  Berkefeld  filters,  and  the  toxin  preserved 
in  fluid  form  with  the  addition  of  0.5  per  cent,  phenol.  As  previously 
mentioned,  the  toxin  rapidly  deteriorates — especially  tetanospasmin — 
and  for  purposes  of  antitoxin  standardization  it  is  usually  preserved  in  a 
dry  state  after  being  precipitated  with  ammonium  sulphate.  The  yel- 
lowish, crystalline  masses  are  readily  soluble  in  water  or  salt  solution, 
and  should  be  used  immediately  after  solution  takes  place.  The  strength 
of  the  toxin  is  determined  by  injecting  increasing  amounts  into  white 
mice  or  350-gram  guinea-pigs. 

1  Jour.  Med.  Research,  1915,  33,  239. 


244  ANTITOXINS 

Immunizing  the  Animals. — According  to  Park,  the  "  horses  receive 
5  c.c.  as  the  initial  dose  of  a  toxin,  of  which  1  c.c.  kills  250,000  grams  of 
guinea-pig,  and  along  with  this  twice  the  amount  of  antitoxin  required 
to  neutralize  it.  In  five  days  this  dose  is  doubled,  and  then  every  five 
to  seven  days  larger  amounts  are  given.  After  the  third  injection  the 
antitoxin  is  omitted.  The  dose  is  increased  at  first  slowly  until  appre- 
ciable amounts  of  antitoxin  are  found  to  be  present,  and  then  as  rapidly 
as  the  horses  can  stand  it,  until  they  support  700  to  800  c.c.  or  more  at  a 
time.  This  amount  should  not  be  injected  in  a  single  place,  or  severe 
local  and  perhaps  fatal  tetanus  may  develop." 

Collecting  the  Serum. — The  horses  are  bled,  and  the  serum  is  col- 
lected under  strict  aseptic  precautions,  in  a  manner  similar  to  the  col- 
lection of  antidiphtheric  serum.  The  serum  should  be  clear  and  free 
from  blood,  and  should  be  proved  sterile  by  cultural  tests.  It  may  be 
preserved  in  the  liquid  state  by  adding  0.5  per  cent,  of  phenol  or  0.4 
per  cent,  of  tricresol. 

Standardizing  the  Serum. — The  official  immunity  unit  of  tetanus 
antitoxin  of  the  United  States  Government  is  based  largely  upon  the 
work  of  Rosenau  and  Anderson.  These  investigators,  together  with  a 
Committee  of  the  Society  of  American  Bacteriologists,  have  defined  the 
unit  of  tetanus  antitoxin  to  be  "ten  times  the  least  amount  of  serum  neces- 
sary to  save  the  life  of  a  350-gram  guinea-pig  for  ninety-six  hours  against 
the  official  test  dose  of  a  standard  toxin.  This  test  dose  consists  of  100 
minimal  lethal  doses  of  a  precipitated  and  dried  toxin,  tested  out 
against  350-gram  pigs,  and  preserved  in  the  Hygienic  Laboratory, 
from  where  it  is  sent  to  various  antitoxin  plants  for  the  purpose  of  secur- 
ing a  uniform  method  and  unit  of  standardization. 

In  standardizing  tetanus  antitoxin,  the  L+  dose  of  toxin  is  em- 
ployed. A  standard  toxin  and  an  antitoxin,  arbitrary  in  their  first 
establishment,  are  preserved  in  the  Hygienic  Laboratory,  and  are  kept 
constant  by  making  frequent  tests  one  against  the  other.  In  determin- 
ing the  L+  dose,  increasing  amounts  of  toxin  are  mixed  with  a  constant 
amount  of  antitoxin  equal  to  one-tenth  of  an  immunity  unit,  and  in- 
jected into  350-gram  pigs.  The  L_|.  dose  must  contain  just  enough 
toxin  to  neutralize  this  amount  of  antitoxin  and  kill  a  pig  in  four  days. 
This  L+  dose  of  toxin  is  sent  out  by  the  Hygienic  Laboratory  to  those 
interested,  commercially  or  otherwise,  in  the  manufacture  of  antitoxin 
for  purposes  of  standardization. 

For  determining  the  strength  of  an  unknown  serum  a  large  number 
of  mixtures  are  made,  each  containing  the  L+  doses  of  the  toxin  and 


ANTIDYSENTERIC    SERUM 


245 


increasing  quantities  of  antitoxin.  The  measurements  are  made  with 
accurate  volumetric  pipets,  and  the  total  volume  brought  up  to  4  c.c. 
with  sterile  salt  solution  in  order  to  equalize  concentration  and  pressure. 
The  mixtures  are  allowed  to  stand  at  room  temperature  for  an  hour,  and 
are  then  injected  subcutaneously  into  350-gram  pigs.  This  method 
of  titrating  the  antitoxin  is  shown  in  the  following  example  from  Rosenau 
and  Anderson: 

TABLE  2.— METHOD  OF  TITRATING  TETANUS  ANTITOXIN 


No.  OF 
PIG 

WEIGHT  OF 
PIG  IN  GRAMS 

SUBCUTANEOUS  INJECTION  OF 
A  MIXTURE  OF  — 

TIME  OF  DEATH 

Toxin  (Test 
Dose) 

Antitoxin 

1            

360 
350 
350 
360 
350 

Gram 
0.0006 
0.0006- 

0.0006 
0.0006 
0.0006 

C.c. 

0.001 
0.0015 
0.002 
0.0025 
0.003 

Two  days  four  hours 
Four  days  one  hour 
Symptoms 
Slight  symptoms 
No  symptoms 

2          

3  

4  
5  

In  this  series  the  animal  receiving  0.0015  c.c.  of  antitoxin  died  in 
approximately  four  days;  this  amount  of  serum,  therefore,  represents 
•^  of  one  unit. 

BOTULINUS  ANTITOXIN 

The  nature  of  the  botulinus  poison  has  previously  been  described. 
Wassermann  has  recently  immunized  horses  against  this  toxin,  and  the 
antitoxin  shows  unmistakable  value  in  animal  experiments,  although 
it  has  not  been  employed  frequently  enough  in  this  form  of  poisoning 
in  human  beings  to  prove  its  value. 


ANTIDYSENTERIC  SERUM 

The  Kruse-Shiga  type  of  dysentery  bacillus  has  been  shown  to  pro- 
duce varying  amounts  of  a  soluble  toxin;  and  antiserums,  which  are 
partly  antitoxic  and  partly  bactericidal  in  nature,  have  been  prepared, 
and  have  apparently  yielded  good  therapeutic  results  in  the  hands  of 
several  observers.  Potent  antiserums  for  the  Flexner  type  of  bacillus 
and  for  various  strains  isolated  from  the  feces  of  cases  of  infantile  ileo- 
colitis  have  not  been  produced.  Even  a  virulent  strain  of  the  dysentery 
bacillus  does  not  produce  true  soluble  toxins  in  a  manner  comparable 


246 


ANTITOXINS 


to  those  produced  by  tetanus  and  diphtheria.  Potent  toxins  are  sel- 
dom secured  with  less  than  two  to  three  weeks'  incubation,  and  fresh 
cultures  of  whole  or  autolyzed  bacilli  are  likewise  quite  too  toxic,  in- 
dicating that  although  a  soluble  toxin  may  be  produced,  considerable 
endotoxin  is  also  present  in  the  bacilli. 

Antidysenteric  serum  has  very  little  prophylactic  value,  but  in  in- 
dividual cases  it  frequently  exerts  a  curative  action,  and  should  be 
available  for  use  in  institutions  and  armies  when  dysenteric  infection 
is  prevalent. 

The  older  investigators,  such  as  Kruse  and  Shiga,  produced  anti- 
serums  by  immunization  with  whole  bacilli.  Later  Kraus  and  Doerr 
prepared  antitoxic  serums  with  the  toxin  alone.  At  the  present  time 
the  evidence  would  seem  to  indicate  that  the  best  serums  are  prepared 
by  injecting  both  toxins  and  bacilli,  producing  a  serum  that  is  essen- 
tially antitoxic  and  bactericidal  in  action. 

Culture. — Young  and  healthy  horses  are  best  adapted  for  immuniza- 
tion. Two  methods  may  be  followed:  (1)  Immunization  with  toxin 
or  (2)  with  young  cultures  of  whole  bacilli.  As  previously  mentioned, 
investigations  have  tended  to  show  that  the  most  potent  serums  are 
secured  by  using  mixtures  of  both  toxin  and  microorganisms. 

Several  strains  of  dysentery  bacilli  should  be  used,  in  order  that  a 
polyvalent  serum  may  be  prepared.  Cultures  should  be  grown  for  two 
weeks  at  37°  C.,  in  alkaline  broth  similar  to  that  used  for  preparing  diph- 
theria toxin;  this  should  be  neutralized  to  phenolphthalein,  and  7  c.c. 
normal  soda  solution  to  a  liter  added.  The  minimal  lethal  dose  of  the 
mixed  unfiltered  cultures  is  determined  by  giving  young  rabbits  increas- 
ing doses  intravenously,  in  order  to  obtain  a  guide  as  to  the  proper  dose 
for  immunization.  Fatal  doses  produce  severe  diarrhea  and  paralysis 
of  the  extremities,  with  rapid  loss  in  weight.  Rabbits  and  horses  are 
quite  susceptible  to  the  toxin;  guinea-pigs  and  mice  are  more  resistant. 

TABLE  3.— METHOD  OF  DETERMINING  THE  MINIMAL  LETHAL  DOSE 
OF  DYSENTERY  CULTURE 


No. 

WEIGHT,  GRAMS 

DOSE  IN  C.c. 

RESULT 

1.  . 

710 

0.025 

No  symptoms 

2  

690 

0.05 

No  symptoms 

3  

695 

0.1 

Diarrhea.     Recovered 

4 

690 

02 

Death  third  day 

5 

700 

0.3 

Death  second  to  third  day 

ANTIDYSENTERIC    SERUM  247 

In  this  instance  the  minimal  lethal  dose  was  0.2  c.c.  and  subsequent 
cultures  of  the  same  strains,  grown  under  similar  conditions,  showed  this 
dose  to  remain  quite  constant. 

It  is  good  practice  to  keep  the  cultures  growing  during  the  entire 
time  of  immunization.  Cultures  may,  however,  be  grown  for  three 
weeks,  filtered  through  porcelain,  and  with  the  addition  of  0.5  per  cent, 
phenol,  the  toxin  preserved  for  long  periods  of  time.  The  minimal 
lethal  dose  of  such  a  toxin  is  determined  in  the  manner  directed  above. 

Immunizing  the  Animals. — Since  horses  are  quite  susceptible,  the 
initial  dose  of  unfiltered  and  unheated  culture  should  not  be  larger 
than  the  minimal  lethal  dose  for  a  young  rabbit.  The  dosage  is  grad- 
ually increased,  and  the  injections  are  given  subcutaneously  for  from 
four  to  six  months,  after  which  several  injections  of  from  300  to  350  c.c. 
may  be  given  intravenously  at  one  time.  If  at  any  time  diarrhea  and 
other  symptoms  of  toxemia  are  well  marked,  subsequent  doses  should 
be  smaller  and  should  be  given  at  longer  intervals  until  a  higher  immu- 
nity is  produced. 

Instead  of  using  bouillon  cultures,  young  agar  cultures  may  be  used, 
the  bacilli  being  grown  for  seventy-two  hours,  and  one-tenth  of  an  ordi- 
nary slant  being  given  as  the  first  dose.  The  early  doses  are  heated  to 
60°  C.  for  an  hour  and  injected  subcutaneously;  the  later  doses  consist 
of  cultures  washed  from  30  to  40  tubes,  and  are  given  intravenously. 

Flexner  and  Amoss  Method  for  Rapid  Production  of  Antidysenteric 
Serum.1 — By  this  method  potent  antidysenteric  sera  can  be  safely 
prepared  in  the  horse  by  the  method  of  three  successive  intravenous 
injections  of  living  cultures  or  toxin  with  intervening  rest  periods  of 
seven  days;  and  effective  serum  for  therapeutic  purposes  may  be  pre- 
pared in  about  ten  weeks.  By  inoculating  alternately  living  bacilli 
belonging  to  the  Shiga  and  Flexner  groups  a  polyvalent  serum  of  high 
titer  may  be  secured  which  should  be  suitable  for  the  serum  treatment 
of  acute  bacillary  dysentery,  irrespective  of  the  particular  strain  or 
strains  of  the  dysentery  bacillus  causing  the  infection. 

Cultures  are  grown  upon  agar-agar  slant  surfaces  in  tubes  15x160 
mm.  in  size  for  twenty-four  hours,  and  the  growth  in  each  tube  suspended 
in  2  c.c.  of  salt  solution.  Horses  are  injected  intravenously;  the  first 
dose  is  1  c.c.  of  the  suspension  of  Flexner  bacilli  after  heating  to 
60°  C.  for  thirty  minutes;  on  each  of  the  following  two  days  5  c.c.  of 
heated  suspension  are  usually  given,  followed  by  a  rest  of  seven  days, 
when  living  cultures  are  injected.  The  temperature  is  taken  daily  and 
1  Jour.  Exper.  Med.,  1915,  21,  515. 


248 


ANTITOXINS 


used  as  an  index  of  the  reaction  and  subsequent  doses.  With  the  living 
bacilli  the  doses  injected  on  each  of  three  days  in  succession  are  four, 
ten,  and  thirty  loopfuls  suspended  in  salt  solution.  Flexner  and  Shiga 
bacilli  are  inoculated  alternately  on  three  successive  days,  with  inter- 
vening rest  intervals  of  seven  days,  the  doses  being  chosen  so  as  to  pro- 
duce a  sharp  febrile  reaction  which  subsides  in  twenty-four  hours.  At 
the  end  of  eight  to  ten  weeks'  immunization  the  serum  contains  immune 
agglutinins  and  a  well-marked  degree  of  antibacterial  and  antitoxic 
value. 

Collecting  and  Testing  the  Serum. — After  three  or  four  months  a 
trial  bleeding  should  be  made  and  the  serum  tested  as  follows :  the  mini- 
mal lethal  dose  of  a  culture  is  determined  and  ten  times  this  amount 
placed  in  a  series  of  tubes  or  syringes  with  increasing  doses  of  serum; 
the  total  quantity  of  injection  is  made  up  to  4  c.c.  with  sterile  salt  solu- 
tion. The  mixtures  are  set  aside  for  one  hour  at  35°  C.  and  injected  in- 
travenously in  young  rabbits.  The  animals  are  to  be  observed  for  at 
least  five  days  for  diarrhea,  paralysis,  and  loss  in  weight.  For  determin- 
ing the  antitoxic  value  a  toxin  is  prepared  by  cultivating  the  bacilli 
in  sugar-free  broth  containing  calcium  carbonate  for  three  days;  the 
bacilli  are  then  killed  with  ether;  the  ether  is  removed  and  the  culture 
filtered  through  hard  paper  or  a  Berkefeld  filter  and  the  toxin  kept  in 
the  refrigerator. 

TABLE  4.— METHOD  OF  TESTING  ANTIDYSENTERIC  SERUM  (KRUSE- 

SHIGA) 


No. 

WEIGHT, 
GRAMS 

CULTURE, 
0.2  C.c.  M.  L.  D. 
C.c. 

SERUM,  C.c. 

RESTTW 

1 

600 

2.0 

0.00025 

Died,  second  day 

2 

610 

2.0 

0.0005 

Died  third  day 

3 

615 

2.0 

0.001 

Diarrhea  recovered 

4             

590 

2.0 

0.002 

Diarrhea  paralysis 

5             

600 

2.0 

0.004 

No  symptoms 

6  

590 

2.0 

0.006 

No  symptoms 

In  this  instance  0.004  c.c.  of  serum  was  sufficient  to  protect  young 
rabbits  against  10  fatal  doses  of  culture,  and  demonstrated  that  it  is 
possible  to  secure  a  fairly  potent  serum  against  the  toxins  of  the  Kruse- 
Shiga  microorganism. 

According  to  Todd,  if  the  antiserum  is  given  at  least  one-half  hour 
after  administering  the  culture,  it  will  protect  the  rabbit.  If  given 
twenty-four  hours  later,  it  affords  no  protection.  Similarly,  the  mix- 
tures of  culture  and  serum  must  not  be  injected  immediately  after  mix- 


ANTISTAPHYLOCOCCUS   SERUM  249 

ing,  as  the  results  are  more  irregular  than  if  they  are  allowed  to  stand  for 
one-half  to  one  hour  before  injecting. 

If  the  trial  bleeding  shows  a  satisfactory  serum,  the  horse  is  bled 
aseptically,  as  was  previously  described,  and  the  serum  is  separated  and 
preserved  with  0.5  per  cent,  phenol  in  quantities  of  10  c.c.  in  sterile 
containers.  As  there  is  no  official  immunity  unit,  the  serum  is  admin- 
istered in  doses  of  from  5  to  10  c.c.  until  a  therapeutic  effect  is  secured. 


ANTISTAPHYLOCOCCUS  "SERUM 

Both  Staphylococcus  pyogenes  aureus  and  S.  pyogenes  albus  have 
been  shown  to  produce  certain  soluble  toxins,  such  as  a  leukocidin  and 
a  hemolysin,  which  are  partly  responsible  for  the  tissue  destruction  and 
symptoms  that  accompany  these  infections.  Severe  staphylococcus 
infection  is  probably  due  in  part  to  the  paralyzing  effect  and  actual  de- 
structive action  of  the  leukocidin  upon  the  leukocytes,  preventing,  for 
the  time  being,  the  walling-off  of  the  lesion  and  effectual  phagocytosis. 
Antistaphylococcus  serums  have  been  shown  to  counteract  the  action 
of  the  leukocidin  and  the  hemolysin,  and  may  be  useful  in  the  treatment 
of  severe,  spreading,  or  metastatic  staphylococcus  infections. 

According  to  Neisser  and  Wechsberg,  during  staphylococcus  dis- 
ease an  antihemotoxin  is  produced  against  the  hemotoxin  of  the  cocci; 
later  Bruck,  Michaelis,  and  Schulze  attempted  to  show  that  a  demon- 
stration of  this  antistaphylolysin  in  the  serum  may  be  regarded  as  evi- 
dence of  a  staphylococcus  infection. 

Preparation  of  Antistaphylococcus  Serum. — For  immunization  pur- 
poses several  different  cultures  of  the  Staphylococcus  aureus  should  be 
used,  in  order  that  the  antiserums  may  be  polyvalent.  Goats  or  horses 
may  be  employed.  Cultures  may  be  grown  on  neutral  agar  for  forty- 
eight  hours,  and  an  emulsion,  equivalent  to  half  an  agar  slant,  heated  to 
60°  C.  for  one  hour  and  injected  subcutaneously  in  an  adult  goat.  If 
10  different  strains  are  used,  a  four-millimeter  loopful  from  each  culture, 
emulsified  in  5  c.c.  of  sterile  salt  solution,  will  be  about  the  proper  dose 
for  the  first  injection.  ^  Subsequent  doses  are  given  at  intervals  of  a 
week,  and  are  rapidly  increased  in  size  until  full,  living,  unheated  cul- 
tures are  injected  intravenously  without  harm  to  the  animal.  The 
serum  may  be  tested  by  determining  its  content  of  antilysin  or  of  bac- 
teriotropin.  Complement-fixation  tests  are  occasionally  useful  for 
obtaining  an  insight  into  the  quantity  of  bacteriolysin  present. 

Technic  of  the  Antilysin  Test. — The  object  of  this  test  is  to  determine 


250 


ANTITOXINS 


the  amount  of  antihemolysin  present  in  a  serum,  which  is  dependent  on 
the  amount  of  serum  necessary  to  protect  the  red  blood-cells  of  rabbits 
against  a  solution  of  the  staphylolysin. 

(a)  Staphylolysin. — This  is  prepared  by  growing  a  known  hemolysin- 
producing  staphylococcus  in  slightly  alkaline  broth  for  three  weeks, 
filtering  through  a  Berkefeld  filter,  and  preserving  the  filtrate  with  0.5 
per  cent,  phenol  in  the  refrigerator. 

(6)  Rabbit  Blood. — Remove  2  or  3  c.c.  of  blood  from  the  ear  of  a  rab- 
bit and  place  in  5  c.c.  of  a  1  per  cent,  sodium  citrate  in  normal  salt  solu- 
tion. Wash  the  corpuscles  three  times,  and  make  up  in  a  1  per  cent, 
suspension  (dose  1  c.c.)  or  up  to  the  original  volume  of  blood  (dose,  1 
drop). 

(c)  Patient's  Serum. — The  serum  is  inactivated  by  heating  to  56°  C. 
for  half  an  hour. 

(d)  Control  Serum. — As  every  normal  serum  contains  a   certain 
amount  of  antilysin,  it  is  necessary  to  use  a  normal  control  serum. 
Normal  horse  serum,  dried  in  vacuo  to  prevent  deterioration,  and  freshly 
dissolved  for  each  test  in  10  volumes  of  sterile  distilled  water  or  salt 
solution,  has  been  advocated  by  Bruck,  Michaelis,  and  Schulze. 

(e)  The  Test. — It  is  first  necessary  to  titrate  the  staphylococcus  fil- 
trate, to  ascertain  the  amount  of  lysin  present.     This  is  accomplished 
according  to  the  following  scheme: 

TABLE  5.— METHOD  OF  TITRATING  STAPHYLOLYSIN 


AMOUNT  OP 
STAPHYLOLYSIN 
FILTRATE 

RABBIT 
BLOOD 
ONE  PER  CENT. 

NORMAL 
SALT  SOLUTION 

RESULT  OF  HEMOLYSIS  AFTER  Two 
HOURS  AT  37°  C.  AND  TWENTY- 
FOUR  HOURS  IN  REFRIGERATOR 

0.005  c.c. 

1  C.C. 

q.  s.  2  c.c. 

No  hemolysis 

0.01    c.c. 

1  C.C. 

q.  s.  2  c.c. 

No  hemolysis 

0.02    c.c.  .  . 

1  C.C. 

q.  s.  2  c.c. 

Slight  hemolysis 

0.05    c.c.  .  . 

1  C.C. 

q.  s.  2  c.c. 

Marked  hemolysis 

0.1      c.c.  .    .. 

1  C.C. 

q.  s.  2  c.c. 

Complete  hemolysis 

0.2      c.c  

1  C.C. 

q.  s.  2  c.c. 

Complete  hemolysis 

0.5      c.c. 

1  C.C. 

q.  s.  2  c.c. 

Complete  hemolysis 

1.0      c.c. 

1  C.C. 

Complete  hemolysis 

In  this  test  0.1  c.c.  is  the  smallest  amount  of  lysin  that  can  completely 
hemolyze  the  given  quantity  of  erythrocytes,  and  is  taken  as  the  unit 
for  the  second  part  of  the  test. 

The  lytic  dose  of  filtrate  just  determined  is  now  placed  in  a  series  of 
small  test-tubes,  with  increasing  doses  of  serum  to  be  tested  and  a  con- 
stant dose  of  corpuscles. 


PRODUCTION   OF  ANTIVENIN 


251 


TABLE  6.— METHOD  OF  TITRATING  ANTISTAPHYLOLYSIN  IN  A  SERUM 


AMOUNT  OP 
FILTRATE 

INACTIVATED 
SERUM 

1  PER  CENT. 
RABBIT 
CORPUSCLES 

NORMAL  SALT 
SOLUTION 

READINGS  AFTER  INCUBATION  AT  37°  C. 

FOR  TWO  HOURS  AND  TWENTY-FOUR 

HOURS  IN  THE  REFRIGERATOR 

0.     C.C  

0     c.c. 

0.001  c.c. 
0  005  c.c. 

1  C.C. 

Ice 

q.  s   2  c.c. 

q    S    2  C  C 

Complete  hemolysis 
Slight  inhibition  of  hemolysis 

0     c.c. 

0.01    c.c. 

Ice 

Q    S    2  C  C 

IVlarked  inhibition  of  hemolvsis 

0.    c.c  
0.    c.c  
0.    c.c  

0.05    c.c. 
0.1      c.c. 
0.2      c.c. 

Ic.c. 
1  c.c. 
1  c.c. 

q.  s  2  c.c. 
q.  s  2  c.c. 
q.  s  2  c.c. 

Complete  inhibition  of  hemolysis 
Complete  inhibition  of  hemolysis 
Complete  inhibition  of  hemolysis 

In  this  instance  0.05  c.c.  of  the  patient's  serum  was  sufficient  com- 
pletely to  neutralize  the  lysin. 

A  similar  test  is  carried  out  with  normal  horse  serum.  The  antilytic 
dose  of  this  serum  is  taken  as  1,  and  the  patient's  serum  is  compared 
with  this  unit.  For  example,  if  0.1  c.c.  of  normal  horse  serum  was  suf- 
ficient to  neutralize  the  lysin  in  this  experiment,  then  the  antilysin  value 
of  the  patient's  serum  is  2. 

According  to  Arndt  and  others,  a  high  antilysin  content  of  a  serum 
is  to  be  regarded  as  indicating  a  staphylococcic  infection,  even  if  it  is 
impossible  to  establish  fixed  limits  for  the  values. 


PRODUCTION  OF   ANTIVENIN 

Snake  venom  contains  two  toxins,  one  being  largely  neurotoxic  and 
producing  paralysis  of  the  respiratory  centers,  and  the  other  being  hemo- 
toxic  and  irritant,  and  producing  local  necrosis  of  tissues,  hemolysis, 
etc.  In  venom  poisoning  the  neurotoxic  effect  is  most  dangerous.  Largely 
as  the  result  of  the  work  of  Calmette  and  Fraser  an  antivenin  has  been 
prepared  that  is  capable  of  counteracting  the  neurotoxic  action  not  only 
of  cobra  venom,  but  to  a  lesser  extent  of  other  venoms  as  well.  These 
serums,  however,  appear  to  have  no  effect  or  but  very  little  upon  the 
irritant  toxins.  In  the  poisonous  American  snakes,  such  as  the  rattler, 
moccasin,  and  copperhead,  the  effects  of  the  irritant  toxins  are  largely 
in  evidence,  and  satisfactory  antiserums  for  these  venoms  have  not 
been  prepared  (McFarland). 

In  preparing  antivenins  the  toxins,  since  they  are  thermolabile, 
must  be  used  unheated;  subcutaneous  injections  are  usually  followed  by 
extensive  sloughing,  and  although  a  certain  amount  of  immunity  may 
be  induced  in  the  horse  by  intravenous  injection,  there  is  apparently  no 
protection  against  the  local  action  of  the  toxins. 


252  ANTITOXINS 

Preparation  of  Antivenin. — According  to  Calmette,  horses  may  be 
immunized  by  giving  them  weekly  subcutaneous  injections  of  gradually 
increasing  doses  of  cobra  venom,  heated  to  70°  C.,  for  an  hour,  which 
precipitates  the  irritant  toxins  without  injuring  the  neurotoxin.  The 
initial  dose  is  usually  0.01  gram,  gradually  increased  until,  by  the  end  of 
four  months,  4  grams  may  be  given  at  a  single  dose.  The  serum  is  then 
tested  by  mixing  increasing  doses  with  the  minimal  lethal  dose  for  a 
young  rabbit,  and  injecting  the  mixtures  intravenously  into  a  series  of 
rabbits. 

Since  the  neurotoxin  may  prove  dangerous  in  any  case  of  snake-bite, 
antivenin  may  be  given  to  advantage,  although  the  local  pain  and  ne- 
crosis are  not  relieved  by  the  serum. 


PRODUCTION  OF  POLLEN  ANTITOXIN 

The  pollen  of  certain  plants  is  markedly  toxic  for  susceptible  in- 
dividuals. In  America  the  pollen  of  the  golden-rod  and  of  rag  weed 
frequently  produce  a  syndrome  of  distressing  symptoms  known  as 
"  autumnal  catarrh."  The  onset  and  character  of  the  symptoms  of 
pollen  intoxication  are  strongly  suggestive  of  an  anaphylactic  reaction. 
Dunbar  has  studied  pollen  toxins  quite  extensively,  and  considers  them 
the  etiologic  factor  in  the  production  of  hay-fever. 

Pollen  antitoxin  has  been  prepared  by  immunizing  susceptible  horses, 
the  toxin  being  isolated  by  mixing  the  ground  pollen  with  5  per  cent, 
sodium  chlorid  solution  and  0.5  per  cent,  phenol  at  37°  C.  for  ten  hours. 
In  the  form  of  a  proteid,  it  is  then  precipitated  by  adding  eight  to  ten 
volumes  of  96  per  cent,  alcohol,  dissolving  the  resultant  white  precipi- 
tate in  physiologic  salt  solution  (Citron). 


THE  MEASURE  OF  ANTITOXINS 

Antitoxin  Unit. — A  unit  is  the  definite  measure  of  antitoxin  in  any 
serum  or  solution  that  will  neutralize  a  certain  amount  of  toxin.  As  pre- 
viously stated,  the  United  States  Government  has  established  a  definite 
unit  for  the  standardization  of  diphtheria  and  tetanus  antitoxins,  and 
frequently  examines  the  serums  made  by  various  licensed  manufacturers. 
Officers  of  the  Public  Health  and  Marine-Hospital  Service  purchase 
from  reliable  pharmacists  several  grades  of  antitoxins  made  by  each 
manufacturer,  which  are  then  sent  to  the  Hygienic  Laboratory  at  Wash- 
ington, where  they  are  tested  for  potency,  freedom  from  contamination 
by  bacteria,  chemical  poisons,  especially  tetanus  toxin,  and  for  excessive 


PRACTICAL   APPLICATION 


253 


amounts  of  preservative.  Delinquencies  are  reported  immediately,  and 
steps  are  taken  to  withdraw  that  particular  lot  of  serum  from  the  market. 

A  unit  of  diphtheria  antitoxin  may  be  defined  as  the  "amount  of  anti- 
toxin that  will  just  neutralize  100  minimal  fatal  doses  of  toxin  for  a  250- 
gram  guinea-pig" 

A  unit  of  tetanus  antitoxin  may  be  defined  as  the  "amount  of  antitoxin 
which  will  just  neutralize  1000  minimal  fatal  doses  of  toxin  for  a  350- 
gram  guinea-pig." 

The  standardization  of  these  serums  is  useful  as  a  guide  to  their  ad- 
ministration, especially  when  given  for  prophylactic  purposes,  where 
experience  has  taught  that  so  many  units  usually  confer  protection;  it 
also  serves  for  purposes  of  record.  In  the  treatment  of  diphtheria  and 
tetanus,  however,  the  serums  are  usually  given  until  a  therapeutic  effect 
is  noted,  regardless  of  the  number  of  units  administered.  If  it  were 
possible  to  determine  quickly  and  accurately  the  amount  of  toxin  in  a 
given  patient,  then  neutralization  could  be  accomplished  along  the  same 
lines  that  make  this  possible  in  the  test-tube.  The  indications  are  to 
administer  at  once  sufficient  antitoxin  to  neutralize  all  the  toxin,  giving 
subsequent  doses  large  enough  to  overcome  the  toxin  as  it  is  produced 
until  the  focus  of  infection  is  removed. 

Antitoxin  should  be  kept  in  a  cold  place  and  protected  from  air  and 
light.  When  this  is  done,  they  usually  do  not  deteriorate  more  than  30 
per  cent,  of  their  original  strength,  and  often  much  less,  within  a  year. 
All  manufacturers  place  a  larger  number  of  units  in  the  container  than 
the  label  calls  for,  in  this  way  allowing  for  the  gradual  loss  in  strength  up 
to  the  date  specified  on  the  label.  According  to  Park,  the  antitoxin  in 
old  serum  is  just  as  effective  as  that  in  fresh  serum,  except  that  there  is 
less  of  it. 

PRACTICAL  APPLICATION 

The  employment  of  antitoxic  serums  both  in  prophylaxis  and  in  the 
treatment  of  infection,  is  considered  in  greater  detail  in  the  chapter  on 
Passive  Immunization  and  Serum  Therapy. 


CHAPTER  XV 
FERMENTS  AND  ANTIFERMENTS 

Bacterial  Ferments. — In  addition  to  the  toxins,  most  bacteria  possess 
certain  ferments  or  enzymes  that  may  play  an  important  role  in  the  pro- 
cesses of  disease.  After  toxins  have  destroyed  body-cells,  proteolytic 
ferments,  partly  derived  from  the  bacteria,  aid  in  their  digestion  and 
produce  a  homogeneous  puriform  substance.  A  similar  condition  may 
be  demonstrated  in  vitro  in  cultures  of  Bacillus  subtilis  on  coagulated 
blood-serum  media  when  the  entire  tube  of  medium  is  quickly  liquefied 
into  a  creamy  fluid  resembling  pus.  The  action  of  the  proteolytic  fer- 
ment is  also  demonstrated  in  the  liquefaction  of  gelatin. 

Pyocyanase  is  a  ferment  that  is  capable  of  digesting  other  bacteria, 
such  as  Bacillus  anthrax,  Bacillus  diphtherise,  staphylococci,  and  strep- 
tococci. It  has  been  asserted  that  the  injection  of  this  ferment  is 
followed  by  an  increased  resistance  to  infection.  Some  years  ago  pyo- 
cyanase  was  manufactured  extensively  and  its  use  advocated  in  the 
treatment  of  local  infections,  the  purpose  being  to  effect  digestion  of 
the  disease-producing  microorganisms.  These  claims  have  not,  however, 
been  substantiated,  and  the  treatment  has  been  generally  abandoned. 

In  addition  to  this  proteolytic  ferment,  bacteria  may  possess  dia- 
static  ferments  capable  of  converting  starches  into  sugars;  inverting 
ferments,  which  may  change  polysaccharids  into  monosaccharids; 
rennin-like  ferments  capable  of  coagulating  milk,  etc.  Not  all  bacteria 
possess  all  these  ferments,  but  a  study  of  them  may  aid  greatly  in  the 
identification  of  the  various  bacterial  species. 

Similarity  Between  Toxins  and  Ferments. — Aside  from  the  definite 
ferments  that  are  in  the  nature  of  secretory  products  of  the  bacteria, 
there  are  many  points  of  resemblance  between  the  toxins,  both  exo- 
toxins  and  endotoxins,  and  the  ferments  or  enzymes.  It  would  seem  that 
the  whole  subject  of  infection  and  immunity  is  becoming  more  and 
more  closely  identified  with  physiologic  chemistry,  especially  with  the 
lipoids  and  lipolytic  ferments.  This  subject  constitutes  today  a  most 
important  field  for  investigation. 

1.  Both  toxins  and  ferments  are  products  of  the  metabolism  of  living 
animal  and  vegetable  cells,  and  may  be  extracellular  (free  enzymes  and 

soluble  toxins)  or  intracellular  (intracellular  enzymes  and  endotoxins). 

254 


SIMILARITY   BETWEEN   TOXINS   AND    FERMENTS  255 

With  diphtheria  bacilli  Kolmer  and  Moshage1  have  shown  that  the  true 
toxin  and  carbohydrate-splitting  ferments  are  independent,  although 
the  ferment-like  carbohydrate-splitting  products  are  most  likely  to  be 
produced  by  toxin-producing  bacilli. 

2.  Both  exhibit  a  latent  period  before  manifesting  their  individual 
activities;    in  general,  the  effect  of  each  is  more  rapid  the  larger  the 
amount  present. 

3.  Both  substances  represent  a  method  or  means  by  which  the  or- 
ganism attempts  to  modify  its  environment  and  render  the  surroundings 
suitable  for  its  nutrition  and  growth. 

4.  Both  show  a  strong  affinity  for  their  substratum,  and  first  manifest 
their  activity  by  combining  with  it.     For  example,  fibrin  placed  in 
gastric  juice  at  0°  C.  and  then  repeatedly  washed  in  cold  water  to  remove 
all  traces  of  pepsin  will  undergo  digestion  when  raised  to  body  tempera- 
ture.    Similarly,  if  red  corpuscles  are  placed  in  fresh  tetanus  toxin  at 
0°  C.  for  an  hour,  washed  repeatedly  with  cold  normal  saline  solution, 
and  then  raised  to  37°  C.,  hemolysis  will  take  place,  indicating  the  pri- 
mary union  of  the  bacterial  hemolysin  or  tetanolysin  with  the  corpuscles. 
In  a  similar  manner  toxins  probably  unite  chemically  with  tissue-cells, 
as  the  toxin  quickly  disappears  from  the  blood  following  its  injection 
and  but  a  small  fraction  can  be  recovered  from  the  excretions.     Further- 
more, the  injection  of  an  emulsion  of  these  cells  into  other  animals  may 
be  followed  by  specific  symptoms  of  intoxication. 

5.  The  activities  of  both  toxins  and  ferments  seem  to  depend  largely 
upon  the  temperature  to  which  they  are  exposed.     For  instance,  in  the 
example  previously  cited,  tetanus  toxin  is  harmless  for  the  frog  until 
the  temperature  of  the  animal  is  raised  to  about  37°  C. 

.  6.  Both  are  usually  affected  by  temperatures  above  70°  C. 

7.  The  one  great  difference,  however,  between  toxins  and  enzymes 
is  the  greater  activity  of  the  latter,  even  very  minute  amounts  of  an 
enzyme  having  the  power  to  split  up  or  decompose  large  quantities  of 
complex  organic  compounds.  An  enzyme  attaches  itself  to  a  substance 
and  absorbs  water;  the  molecule  breaks  down,  the  enzyme  is  liberated, 
and  then  attacks  another  molecule,  this  process  being  repeated  until 
Harge  amounts  of  fermentable  substances  have  been  attacked.  When, 
however,  a  toxin  has  united  with  a  substance  it  loses  its  identity,  and  in 
this  manner  it  follows  the  law  of  multiple  proportions.  This  has  been 
discussed  as  it  relates  to  the  soluble  toxins  of  diphtheria  and  tetanus, 
and  is  likewise  easily  demonstrable  in  the  action  of  tetanolysin  upon 
erythrocytes  of  the  rabbit.  It  is  true  that  a  toxin  may  become  dissoci- 
1  Jour.  Infect.  Dis.,  1916,  19,  28. 


256  FERMENTS   AND    ANTIFERMENTS 

ated  and  attack  another  molecule,  but  this  action  is  different  from  that 
of  an  enzyme,  because  the  molecule  first  attacked  is  not  injured.  How- 
ever, as  Adami  points  out,  the  toxins  may  be  equally  active  in  the  body 
until  arrested  by  antitoxins,  although  experiments  in  vitro  clearly  demon- 
strate the  greater  activity  of  the  ferments. 

8.  Even  in  serum  hemolysis  it  would  appear  that  a  lipolytic  ferment 
is  vitally  concerned.  According  to  Jobling  and  Bull,1  the  end-piece  of 
split  complement  contains  a  lipolytic  ferment.  These  observers  be- 
lieve that  a  parallelism  exists  between  lipase  content  and  complement 
activity  of  the  serums  of  various  animals.  As  will  be  pointed  out  further 
on,  the  complements  possess  many  properties  resembling  those  of  the 
ferments,  and  many  factors  are  being  established  to  show  the  important 
relation  that  lipoids  and  Upases  bear  to  immunologic  processes. 

ANTIFERMENTS 

According  to  some  investigators,  antif erments  are  to  be  found  in  large 
amounts  in  all  normal  serums,  and  are  probably  vitally  concerned  in  the 
processes  of  life  in  preventing  autodigestion.  That  they  may  be  in- 
creased in  number  artificially  by  immunization  up  to  a  certain  limit 
has  been  disputed;  it  is  certain  that  they  never  attain  the  extreme 
amounts  possible  with  the  injection  of  toxins.  This  may  be  due  to  the 
formation  of  anti-anti-enzymes,  produced  by  a  regulating  mechanism 
that  prevents  anti-enzymes  from  accumulating  beyond  a  certain  point 
and  interfering  with  nutrition.  It  is  possible  that  the  body  mechanism 
exerts  a  strict  regulating  effect  between  the  formation  of  enzymes  and 
anti-enzymes.  Furthermore,  when  free  receptors,  such  as  normal 
anti-enzymes,  are  present  hi  the  body-fluids,  the  body-cells  are  not 
stimulated  to  produce  these  anti-enzymes  in  excess,  nor  does  the  pres- 
ence of  the  free  receptors  stimulate  the  cells  to  produce  anti-bodies 
against  their  normal  side-chains. 

Many  investigators  claim  to  have  produced  antiferments  experi- 
mentally. Morgenroth 2  believed  that  he  obtained  a  specific  antirennin 
by  inoculating  goats  with  rennin.  Sachs 3  and  Achaline 4  assert  that  they 
have  produced  specific  antipepsin  or  antitrypsin  by  inoculating  animals 
with  these  ferments.  Antisteapsin  and  antilactase  have  been  prepared 
by  Schutze,5  antityrosinase  by  Gessard,6  and  antiurease  by  Moll.7 

On  the  other  hand,  these  observations  have  not  been  generally  con- 

1  Jour.  Exper.  Med.,  1913,  xvii,  61.         2  Centralbl.  f.  Bakteriol.,  1899,  xxvi,  349. 
3  Fortschr.  d.  Med.,  1902,  20,  425.          4  Ann.  de  1'Inst.  Pasteur,  1901,  xv,  737. 
6  Zeitschr.  f.  Hygiene,  1904,  48,  457;  Deutsch.  med.  Wochen.,  1904,  30,  308. 
6  Ann.  de  1'Inst.  Pasteur,  1901,  15.         7  Hofmeister's  Beitr.,  1902,  2. 


ANTIFERMENTS  257 

firmed.  The  inhibitory  substances  on  trypsin  in  blood-serum,  for  in- 
stance, have  been  ascribed  by  Jobling  and  Petersen1  to  the  presence  of 
compounds  of  the  unsaturated  fatty  acids. 

Antibodies  and  Antiferments. — Elsewhere  has  been  discussed  more 
fully  the  parallelism  that  exists  between  bacterial  antibodies  and 
antiferments.  One  is  impressed  with  the  similarity  of  the  processes 
concerned  in  the  breaking  down  of  food-stuffs  into  simple  substances 
for  assimilation  by  ferments  and  the  destruction  of  bacteria  and  their 
products  by  antibodies.  The  processes  that  occur  when  a  cell  digests 
an  ingested  microbe  by  a  cytase  must  be  similar  to  that  which  occurs 
in  the  digestion  of  any  other  foreign  matter,  and  it  is  but  a  short  step 
to  conceive  that  bacteriolysis  in  the  body  fluids  is  similar  to  the  processes 
concerned  in  the  digestion  of  protein  by  the  body  cells.  The  close 
similarity  that  exists  between  the  toxins  and  the  ferments,  the  anti- 
toxins and  the  antiferments,  complements  and  kinases,  and  the  quantita- 
tive relations  existing  between  a  toxin  and  its  antitoxin,  between  a  fer- 
ment and  its  antiferment — all  these  indicate,  as  Adami  has  pointed  out, 
the  close  parallelism  that  exists  between  toxins  and  cytolysins  and  fer- 
ments of  different  orders  and  grades.  They  would  seem  to  indicate, 
moreover,  that  we  are  probably  dealing  with  one  common  group  of  en- 
zymic  substances  that  act  not  by  physical  contact,  but  by  chemical 
combination. 

Antiferments  in  Disease. — In  this  connection  a  subject  of  consider- 
able interest  is  the  probable  nature  of  the  syphilitic  antibody  so  vitally 
concerned  in  the  Wassermann  reaction.  Although  the  true  nature  of 
this  reaction  is  unknown,  there  can  be  no  doubt  as  to  the  intimate  re- 
lation of  lipoidal  substances  to  the  processes  concerned.  It  is  probable 
that  the  Treponema  pallidum  produces  a  true  antibody,  and  a  second 
body,  in  the  nature  of  a  cellular  reactionary  substance,  which  has  a 
marked  affinity  for  lipoids.  When  the  two  substances  are  mixed  and 
complement  added,  the  latter  is  adsorbed  or  inactivated  to  a  greater  or 
less  degree,  so  that  upon  the  subsequent  addition  of  washed  erythrocytes 
and  its  corresponding  hemolytic  amboceptor  hemolysis  either  does  not 
occur  at  all  or  is  more  or  less  incomplete.  A  similar  "reagin"  is  present 
in  the  blood-serum  of  persons  suffering  with  yaws  and  tuberculous  lep- 
rosy, and  although  its  exact  nature  is  unknown,  its  peculiar  behavior 
toward  the  lipoids  suggests  strongly  that  it  is  a  product  of  the  toxic 
action  of  the  causal  parasite  upon  the  lipoids  of  the  body-cells  and  is  in 
the  nature  of  an  antilipoid.  (See  Wassermann  reaction.) 

1  Jour.  Exper.  Med.,  1914,  xix,  459. 
17 


258  FERMENTS   AND   ANTIFERMENTS 

Jochmann  and  Muller  have  demonstrated  the  presence  of  an  anti- 
ferment  in  the  serum  used  against  leukocytic  ferments  in  diseases  as- 
sociated with  great  destruction  of  the  leukocytes.  Following  these 
observers,  Marcus  Brieger  and  Trebing x  found  that  90  per  cent,  of  the 
patients  suffering  from  carcinoma  or  sarcoma  examined  by  them  showed 
an  increase  of  antitrypsin  in  the  blood.  Von  Bergmann  and  Meyer2 
confirmed  this  observation,  although  they  found  that  a  similar  increase 
also  occurred  in  24  per  cent,  of  non-cancerous  patients.  More  recent 
work  would  indicate  that  the  antitrypsin  may  be  present  in  acute  in- 
fections, such  as  pneumonia,  typhoid  fever,  etc.,  in  chronic  infections, 
such  as  tuberculosis  and  syphilis,  in  exophthalmic  goiter,  and  in  severe 
anemias.  As  previously  mentioned,  Schwartz,3  Suginoto,4  and  Job- 
ling  and  Petersen 5  believe  that  the  antitryptic  influence  of  blood-serum 
is  due  to  the  lipoids,  and  especially  to  the  compounds  of  the  unsaturated 
fatty  acids. 

The  tryptic  ferment  liberated  by  disintegrating  leukocytes  and  con- 
nective-tissue cells  is  largely  responsible  for  the  liquefaction  of  these 
cells  and  the  formation  of  pus,  as  in  abscess  formation  and  autodigestion 
of  infected  surface  wounds.  On  the  other  hand,  an  antitrypsin-like 
substance  tends  to  limit  the  activities  of  the  ferment  and  protect  the 
surrounding  tissues  from  progressive  destruction.  A  deficiency  of  this 
substance  may  account  for  the  rapid  breaking-down  of  infected  glands 
and  of  a  walled-off  tuberculous  lesion,  the  development  of  carbuncles, 
etc.  A  study  of  the  antitryptic  power  of  the  blood  may,  therefore,  prove 
of  value  in  suppurative  processes  and  in  malignant  disease,  and  consider- 
ably influence  a  prognosis. 

Ferments  in  Pregnancy  and  Disease. — It  is  largely  to  the  researches 
of  Abderhalden  and  his  associates  that  we  owe  our  knowledge  of  the 
fact  that  when  food-stuffs  are  introduced  into  the  body  parenterally,  i.  e., 
by  subcutaneous  or  intravenous  injection,  ferments  are  produced  that,  by 
process  of  cleavage  and  reduction,  deprive  them  of  their  individuality. 

For  example,  as  shown  by  Weinland,6  normal  dog  serum  cannot 
reduce  cane-sugar,  whereas  the  serum  of  a  dog  immunized  by  several 
injections  of  this  sugar  is  able  to  reduce  it  in  vitro  by  means  of  a  specific 
ferment  of  the  nature  of  invertin.  Similarly,  normal  serum  is  unable 
to  cleave  edestin  (vegetable  albumin),  whereas  the  serum  of  an  im- 
munized dog  will  split  this  protein  into  simpler  substances. 

1  Berl.  klin.  Wochschr.,  1908,  xlv,  1349.  2  Ibid.,  1908,  xlv,  1673. 

3  Wien.  klin.  Wochschr.,  1909,  xxii,  1151. 

4  Arch.  f.  exper.  Path.  u.  Pharmakol.,  1913,  Ixxii,  374. 

6  Jour,  exper.  Med.,  1914,  xix,  239  and  459.         6  Ztschr.  f.  Biol.,  1907,  279. 


ANTIFERMENTS  259 

After  he  had  proved  experimentally  that  the  animal  organism  is  able 
to  mobilize  ferments  against  foreign  substances,  Abderhalden  next 
took  up  the  question  whether  ferments  are  produced  when  substances 
native  to  the  body  but  foreign  to  the  blood  are  introduced  into  the  cir- 
culation. Having  learned  from  the  researches  of  Veit,  Schmorl,  Weichard, 
and  others  that  during  pregnancy  syncytial  cells  frequently  enter  the 
maternal  circulation,  Abderhalden  used  the  serums  of  pregnant  animals, 
and  found  that  they  contained  a  ferment-like  substance  capable  of 
splitting  placental  peptone  into  amino-acids  and  coagulated  placenta 
into  peptones,  polypeptids,  and  amino-acids. 

It  was  apparently  thus  established  that  the  body  cells  are  harmonic- 
ally attuned  to  one  another,  and  if  new  or  modified  cells  or  their  prod- 
ucts are  brought  into  relation  with  other  cells,  they  are  received  as 
foreign  invaders,  and  their  entrance  is  followed  by  the  production  of 
what  Abderhalden  has  called  "protective  ferments"  ("Abwehrfermente") 
capable  of  bringing  about  their  cleavage  into  simpler  products.  In  this 
manner  the  presence  in  the  circulation  of  some  of  the  body  cells  may 
give  rise  to  the  production  of  these  ferments  if  the  cells  in  question  are 
really  foreign  to  the  blood-plasma  and  other  cells. 

Abderhalden  has  also  stated  that  although  he  was  led  to  make 
these  investigations  on  the  supposition  that  syncytial  elements  were 
present  in  the  blood  of  pregnant  women,  it  is  not  necessary  that  they  be 
constantly  in  the  blood,  for  every  case  of  pregnancy  has  a  complicated 
protein  metabolism  and  there  is  a  general  exchange  of  substances  between 
the  placenta  and  the  maternal  blood  that  permits  the  entrance  into  the 
latter  of  protein  products  that  have  not  been  broken  down  completely 
into  amino-acids,  and  that  cause  the  organism  to  produce  defensive 
proteolytic  ferments. 

In  cancer,  where  the  production  of  new  cells  is  so  marked,  some  of 
these  cells  or  their  products  may  easily  be  swept  into  the  general  circula- 
tion, where  they  act  as  foreign  invaders  and  cause  the  formation  of  pro- 
tective proteolytic  ferments.  It  is  a  noteworthy  fact,  moreover,  that 
the  serum  of  carcinoma  cases  reacts  best  with  carcinoma  cells  and  that 
of  sarcoma  with  sarcoma  cells. 

Similar  ferments  have  been  described  in  other  conditions.  Fauser 
has  demonstrated  that  the  blood-serum  of  dementia  prsecox  patients 
contains  ferments  that  act  on  the  reproduction  glands,  so  that  the  serum 
of  males  reacts  with  testicular  extracts  and  that  of  females  with  ovarian 
extracts.  These  serums  were,  however,  also  found  to  react  with  thyroid 


260  FERMENTS   AND   ANTIFERMENTS 

tissue  and  brain  cortex.  In  general  paresis  reactions  were  obtained  with 
brain  cortex  and  liver,  also  at  times  with  thyroid  gland,  reproductive 
glands,  and  more  rarely  with  kidney. 

Abderhalden  has  found  ferments  for  the  tubercle  bacilli  in  the 
blood-serum  of  tuberculous  persons,  and  they  have  also  been  found 
in  the  blood-serum  of  syphilitics  for  the  Treponema  pallidum,  either 
in  pure  culture  or  in  organs  containing  large  numbers  of  the  parasites; 
Smith1  has  found  a  remarkable  specificity  of  the  ferments  produced 
in  rabbits  following  immunization  with  typhoid  and  paratyphoid  bacilli 
and  cocci. 

Although  these  protective  ferments  are  in  general  similar  to  the  cyto- 
lysins  or  antibodies  produced  during  bacterial  and  protozoan  infections 
capable  of  lysing  or  digesting  their  antigens,  their  exact  nature  and  rela- 
tion to  the  cytolysins  have  not  been  determined.  From  the  evidence 
at  hand  it  would  appear  that  the  ferment-like  cytolysin  is  specifically 
directed  against  the  toxic  portion  of  a  bacterial  cell,  and  the  proteolytic 
ferment  against  the  bacterial  cell  itself.  Recent  work  by  Pearce  and 
Williams2  would  seem  to  indicate  that  the  cytolysins  and  protective 
ferments  are  separate  substances,  but  considerable  additional  experi- 
mental investigation  is  required  to  clear  up  the  point. 

Mechanism  of  the  Abderhalden  Reaction. — Aside  from  the  probable 
clinical  value  of  the  methods  devised  by  Abderhalden  in  the  serum  diag- 
nosis of  pregnancy  and  various  pathologic  conditions,  as  malignancy, 
tuberculosis,  lesions  of  the  nervous  system  and  ductless  glands,  most 
interest  concerns  the  question  of  the  specificity  of  the  ferments  or  anti- 
bodies concerned  and  the  mechanism  of  their  action. 

While  Abderhalden  and  many  of  his  pupils  have  claimed  a  high  degree 
of  specificity  for  the  "protective  ferments"  and  his  pregnancy  reaction, 
claiming  from  the  beginning  that  errors  of  technic  were  largely  respon- 
sible for  the  failure  of  others  to  obtain  satisfactory  results,  the  dialysis 
test  as  now  conducted  is  not  especially  difficult,  and  sufficient  work  has 
been  done  by  other  investigators  who  have  followed  Abderhalden's 
technic  with  great  care  and  exactness  to  give  warrant  to  the  claim  that 
other  factors  aside  from  those  purely  technical  may  be  responsible  for 
the  divergent  and  non-specific  results  obtained. 

As  previously  stated,  Abderhalden  bases  his  theory  concerning  the 
"protective  ferments"  upon  the  specific  digestion  of  a  substrat  by 
specific  ferments,  claiming  that  these  ferments  are  separate  and  dis- 

1  Jour.  Infect.  Dis.,  1916,  18,  14  2  Jour.  Infect.  Dis.,  1914,  xiv,  351. 


ANTIFERMENTS  261 

tinct  antibodies  and  not  to  be  classed  with  the  cytolytic  amboceptors 
or  cytolysins  of  Ehrlich. 

That  the  substrat  in  the  pregnancy  test  is  a  boiled  tissue  would  seem 
to  impair  the  specificity  of  the  reaction,  and,  indeed,  certain  physical 
factors,  as  the  mechanical  state  of  division  of  the  substrat  and  its  facility 
for  acting  as  an  absorbent  in  a  purely  mechanical  capacity,  likewise 
appear  to  be  factors  in  the  reaction  on  the  basis  of  numerous  investi- 
gations showing  that  loose  areolar  placental  tissue  is  frequently  digested 
by  normal  sera  and  various  pathologic  sera  irrespective  of  pregnancy, 
whereas  digestion  of  a  firm  and  compact  tissue  as  that  of  malignant 
tumors  is  much  less  constant.  In  this  connection  the  work  of  de  Waele1 
has  a  bearing,  inasmuch  as  he  found  that  any  agent  which  would  cause 
an  alteration  of  the  physical  state  of  the  serum  globulins  would  cause 
an  intense  Abderhalden  reaction,  concluding  that  the  reaction  depended " 
upon  a  globulinolysis  having  an  origin  in  physical  processes  probably 
analogous  to  the  precipitin  reaction. 

While  immunologic  as  well  as  chemical  and  physical  reactions  are 
more  or  less  dependent  upon  quantitative  factors,  recent  investigations 
by  Flatow2  and  Herzfeld,3  Plaut,4  and  others  show  that  while  specific 
results  may  be  obtained  by  proper  manipulation  of  the  material,  non- 
specific results  in  either  a  negative  or  positive  reaction  may  be  obtained 
with  practically  any  serum,  however  well  controlled,  with  the  same 
material.  These  investigations  are  significant  not  so  much  because  of 
quantitative  factors  alone  as  they  are  by  reason  of  indicating  that  the 
pregnancy  reaction  is  dependent  upon  the  principles  of  mechanical 
absorption  on  the  part  of  the  substrat  of  something  from  the  serum 
followed  by  a  digestive  process,  rather  than  upon  the  simple  digestion 
of  a  specific  substrat  by  a  specific  ferment. 

For  this  conception  of  the  mechanism  of  the  Abderhalden  reaction 
the  investigations  of  Jobling  and  Peterson5  have  been  fundamental  and 
of  great  interest  and  importance.  They  have  shown  that  the  digestive 
power  of  a  serum  is  dependent  upon  non-specific  proteolytic  ferments 
or  proteases  normally  present  and  held  in  check  by  an  antiferment, 
which,  according  to  their  work,  is  believed  to  reside  in  the  unsaturated 
fatty  acids  of  the  serum.  Upon  removal  of  the  antiferment  by  means  of 

1  Ztsch.  f.  Immunitatsf .,  orig.,  1914,  12,  170. 

2  Munch,  med.  Wchnschr.,  1914,  Ixi,  468;  ibid.,  608;  ibid.,  1168. 

3  Biochem.  Ztschr.,  1914,  Ixiv,  103. 

4  Munch,  med.  Wchnschr.,  1914,  Ixi,  238. 

5  Jour.  Exper.  Med.,  1914,  xix,  459  and  480. 


262  FERMENTS   AND    ANTIFERMENTS 

lipoidal  solvents  or  saturation  with  various  organic  and  inorganic 
substances,  as  boiled  tissue,  iodin,  starch,  kaolin,  and  the  like,  protease 
activity  is  released,  followed  by  digestion,  not  of  the  so-called  substrat 
but  of  the  protein  of  the  serum.  Likewise  Plaut,1  Peiper,2  Friedman 
and  Schonfield,3  and  Bronfrenbrenner4  have  obtained  positive  Abder- 
halden  reactions  with  guinea-pig  and  human  sera  not  only  with  placental 
tissue  but  also  with  such  inert  substances  as  kaolin,  starch,  barium  sul- 
phate, chloroform,  etc.  These  studies  would,  therefore,  tend  to  show 
that  the  boiled  placental  tissue  in  Abderhalden's  reaction  is  not  digested, 
but  acts  simply  as  an  absorbent  in  a  purely  mechanical  manner. 

Furthermore,  Heilner  and  Petri5  and  de  Waele6  found  the  ferments 
in  the  blood-serum  so  quickly  after  the  parenteral  introduction  of  the 
protein,  at  intervals  hardly  sufficient  for  the  elaboration  of  new  and 
specific  ferments,  as  to  support  the  theory  that  the  ferments  are  pre- 
formed and  that  the  substrat  serves  to  activate  these  rather  than  bring 
about  the  production  of  new  ferments. 

The  researches  of  Van  Slyke  and  his  associates,7  employing  Van 
Slyke's  method  of  amino  nitrogen  determination,  have  shown  that 
practically  every  serum,  whether  from  a  pregnant  or  non-pregnant 
individual,  showed  protein  digestion  when  incubated  with  placenta 
tissue  prepared  according  to  Abderhalden;  further  evidence  of  non- 
specificity  was  seen  in  the  fact  that  carcinoma  tissue  was  digested 
apparently  to  about  the  same  extent  as  was  placenta.  Hulton,8  working 
with  isolated  and  purified  proteins,  found  no  digestion  with  the  sera  of 
injected  rabbits  in  excess  of  that  which  took  place  with  the  sera  of  normal 
control  animals,  concluding  that  there  is  at  present  no  reason  for  believing 
that  the  normal  hydrolysis  of  the  protein  of  the  body  occurs  in  the  circu- 
lating blood,  but  that  these  metabolic  changes  presumably  belong  to 
the  tissue  cells. 

It  would  appear,  therefore,  that  the  original  theory  of  Abderhalden 
is  untenable  in  that  specific  proteolytic  ferments  in  the  blood  are  not 
produced  during  pregnancy,  and  that  the  Abderhalden  reaction  is  not 
due  to  the  digestion  in  vitro  of  specific  antigen  by  specific  ferments. 

1  Munch,  med.  Wchnschr.,  1914,  Ixi,  238. 

2  Deut.  med.  Wchnschr.,  1914,  xl,  1467. 

3  Berl.  klin.  Wchnschr.,  1914,  li,  348. 

4  Proc.  Soc.  Exper.  Biol.  and  Med.,  1914,  xi,  90. 

5  Munch,  med.  Wchnschr.,  1911,  Ix,  1530. 
6Ztschr.  f.  Immunitatsf.,  orig.,  1914,  xxii,  31. 

7  Jour.  Biol.  Chem.,  1915,  23,  377. 

8  Jour.  Biol.  Chem.,  1916,  25,  163. 


ANTIFERMENTS  263 

On  the  other  hand,  I  am  of  the  opinion  that  the  last  word  has  not  been 
spoken,  and  while  the  Abderhalden  test  is  subject  to  so  much  error  as 
to  greatly  reduce  its  practical  applications,  the  sera  of  pregnant  women 
contain  an  antibody-like  substance  in  such  concentration  as  is  not 
found  in  the  sera  of  males  and  non-pregnant  women.  In  our  own  experi- 
ments with  Asnis  and  Freese,  the  Van  Slyke  method  was  found  to  be 
insufficiently  delicate  for  demonstrating  the  difference  between  the  sera 
of  pregnant  and  non-pregant  persons.  In  our  experiments  with  Wil- 
liams,1 while  reactions  occurred  with  the  sera  of  pregnant  women  and 
tissue  substrats  other  than  placenta,  and  also  with  such  substances  as 
kaolin,  starch,  and  agar,  stronger  reactions  occurred  with  placental 
substrat;  for  this  reason  I  am  of  the  opinion  that  in  pregnancy  serum 
there  are  two  sets  of  proteolytic  ferment-like  substances,  one  normal 
and  non-specific  and  the  second  specific;  while  the  former  may  be  acti- 
vated by  various  non-specific  organic  and  inorganic  substances  re- 
sulting in  the  digestion  of  the  serum,  the  latter  are  activated  by  the 
specific  substrat  alone,  resulting  in  the  digestion  of  not  only  the  serum 
but  also  to  some  extent  of  the  placental  substratum  itself.  Bronfen- 
brenner2  is  of  the  opinion  that  the  apparently  specific  phase  of  the 
Abderhalden  reaction  is  due  to  the  specific  combination  of  the  antigen 
of  the  substratum  with  the  antibody  of  the  serum,  leading  to  a  change 
of  colloidal  conditions  resulting  in  the  removal  of  the  substances  which 
inhibit  digestion. 

1  Amer.  Jour.  Obstet.  and  Dis.  Women  and  Children,  1915,  Ixxii,  No.  1. 

2  Jour.  Lab.  and  Clin.  Med.,  1915,  1,  79. 


264  FERMENTS   AND    ANTIFERMENTS 

FERMENT  REACTIONS 
ANTITRYPSIN  TEST 

Bergmann  and  Meyer1  have  devised  a  test  for  estimating  the  titer 
of  antitrypsin  that  possesses  value  in  determining  the  presence  of  tryp- 
tic  ferment  hi  the  blood-serum  and  in  the  intestinal  and  stomach  con- 
tents. The  test  may  also  prove  of  value  in  making  a  functional  study 
of  the  pancreas.  At  present  it  is  regarded  by  some  as  an  aid  to  the 
diagnosis  of  cancer,  and  it  may  also  be  of  service  in  establishing  the 
diagnosis  and  prognosis  of  suppurative  processes. 

Solution  of  Trypsin. — This  is  made  by  dissolving  0.5  gm.  of  pure 
trypsin  (Griibler)  in  50  c.c.  of  NaCl  solution  and  adding  0.5  c.c.  of  normal 
soda  solution;  make  up  to  500  c.c.  with  physiologic  salt  solution. 

Casein  Solution. — Dissolve  one  gram  of  pure  casein  in  100  c.c.  of 
sodium  hydroxid  solution  with  the  aid  of  gentle  heat.  Neutralize  to 
litmus  with  ^  hydrochloric  acid  solution  and  dilute  with  physiologic 
salt  solution  up  to  500  c.c.  Filter  and  sterilize  in  an  Arnold  sterilizer. 
Preserve  in  the  refrigerator. 

Acetic  Acid  Solution. — To  5  c.c.  of  acetic  acid  (c.  p.),  add  45  c.c. 
of  absolute  alcohol  and  50  c.c.  of  distilled  water. 

The  patient's  serum  must  be  fresh,  and  should  be  diluted  20  times 
with  salt  solution.  Dose,  0.2  c.c. 

Technic. — A  titration  of  the  trypsin  solution  must  precede  the  test 
proper.  Into  each  of  several  small  test-tubes  place  increasing  amounts 
of  trypsin  solution,  as,  for  example,  0.1,  0.2,  0.4,  0.6,  0.8,  and  1.0  c.c. 
Add  2  c.c.  of  the  casein  solution  to  each  tube;  shake  carefully  and  place 
in  an  incubator  or  water-bath  for  half  an  hour  at  50°  C.  Then  add 
three  or  four  drops  of  the  acetic  acid  solution  to  each  tube,  and  observe 
which  tube  first  shows  cloudiness  after  a  few  minutes.  The  tube  con- 
taining the  smallest  amount  of  trypsin  and  which  remains  perfectly 
clear  contains  enough  trypsin  fully  to  digest  the  2  c.c.  of  casein  solution. 

Into  each  of  six  small  test-tubes  now  place  0.2  c.c.  of  the  1  :  20  dilu- 
tion of  the  patient's  serum,  and  increasing  amounts  of  the  trypsin  solu- 
tion, beginning  with  the  completely  digesting  dose,  as  determined  above, 
and  increasing  by  0.1  c.c.  Add  2  c.c.  of  casein  solution  to  each  tube, 
and  bring  all  tubes  to  a  like  volume  by  the  addition  of  normal  salt  solu- 
tion. Shake  gently,  and  incubate  at  50°  C.  for  half  an  hour.  Add 

1  Berl.  klin.  Wochenschr.,  1908,  No.  37. 


ANTITRYPSIN   TEST  265 

several  drops  of  acetic  acid  solution  to  each  tube,  and  again  observe  the 
tube  containing  the  smallest  amount  of  trypsin  in  which  cloudiness  can 
be  seen.  In  this  way  the  amount  of  trypsin  neutralized  by  the  anti- 
trypsin  of  the  serum  is  determined. 

For  example,  in  an  experiment  the  preliminary  titration  showed  that 
0.5  c.c.  of  trypsin  completely  digested  the  casein.  In  the  second  part 
of  the  test  the  lower  limit  of  trypsin  was  this  0.5  c.c.  increased  by  0.1 
c.c.  in  successive  tubes  up  to  1  c.c.  It  is  now  found  that  1  c.c.  of  the 
trypsin  solution  is  required  to  bring  about  the  complete  digestion  of  the 
casein  in  the  presence  of  the  serum,  or  1  c.c.— 0.5  c.c.  =0.5  c.c.,  which  is 
the  amount  of  trypsin  neutralized  by  0.01  c.c.  of  undiluted  serum. 

A  control  experiment  is  conducted  with  the  pooled  serum  of  several 
normal  persons,  and  a  comparison  of  the  value  thus  obtained  shows 
whether  the  antitryptic  power  of  the  serum  tested  is  altered. 

The  method  of  Marcus,1  which  is  a  modification  of  the  method  of 
Muller  and  Jochmann,2  is  described  in  the  laboratory  exercises  on 
Experimental  Infection  and  Immunity. 

ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY' 
Methods. — Two  methods  have  been  devised  by  Abderhalden  for 
the    demonstration   of   the   ferments   in    the    blood-serum    of    preg- 
nancy : 

1.  The   Dialyzation   Method. — Specially   prepared   and   coagulated 
placenta  and  fresh  serum  are  placed  in  a  dialyzing  capsule  so  prepared 
that  it  will  permit  the  passage  of  peptones  and  amino-acids  only.    The 
filled  capsule  is  placed  in  sterile  distilled  water,  and  incubated  for  from 
sixteen  to  twenty-four  hours,  when  the  dialysate  is  tested  by  the  biuret 
or  ninhydrin  test  for  peptones  and  amino-acids.    Under  proper  condi- 
tions the  presence  of  these  substances  indicates  that  the  placental  tis- 
sue has  been  digested  by  the  serum. 

2.  The  Optical  Method. — This  method  is  based  upon  the  same  prin- 
ciple as  the  dialyzation  method.    Into  the  tube  of  a  polariscope  place  a 
solution  of  placental  peptone  and  the  serum  to  be  tested.    Warm  the 
mixture  to  37°  C.,  and  after  an  hour  note  the  degree  of  rotation  and 
record  it;  repeat  this  at  intervals  during  the  following  twenty-four  to 

1  Berl.  klin.  Wochenschr.,  1908,-  No.  4;  1909. 

2  Munch,  med.  Wochenschr.,  1909,  Nos.  29  and  31. 

3  Abderhalden:     Abwehrfermente  des  tierischen  Organisims,   Julius  Springer, 
third  edition,  1913. 


266  FERMENTS   AND   ANTIFERMENTS 

forty-eight  hours.  If  the  serum  contains  a  proteolytic  ferment,  the 
peptone  is  split  into  amino-acids  and  the  degree  of  rotation  increased 
from  0.05°  to  0.5°  and  higher.  This  method  requires  an  expensive  polar- 
iscope,  considerable  practice  in  making  the  observations  and  readings, 
and  is  only  reliable  in  skilful  hands. 

THE  DIALYZATION  METHOD 

Testing  the  Dialyzing  *Shell. — The  quality  of  the  dialyzing  shell 
largely  determines  the  success  of  this  method.  It  must  fulfil  two  re- 
quirements : 

1.  It  must  be  absolutely  non-permeable  for  albumin. 

2.  It  must  be  evenly  permeable  for  the  protein  cleavage  products, 
such  as  peptones,  polypeptids,  and  amino-acids. 

Special  shells  are  made  by  Schleichter  and  Schull,  No.  579a  being 
recommended  at  the  present  time.  The  shells  must  be  of  correct  size, 
and  every  one  must  be  tested  before  being  used.  If  a  shell  allows  un- 
cleaved  protein  to  pass  through,  then  all  reactions  would  react  posi- 
tively regardless  of  the  presence  or  the  absence  of  the  specific  ferment. 
If  the  shell  is  too  thick  and  too  tight,  and  prevents  the  passage  of  pep- 
tones and  ammo-acids,  then  all  reactions  would  be  negative,  even  though 
the  ferment  were  present  in  the  serum  and  had  digested  the  placental 
protein.  Accordingly,  each  shell  must  be  tested  and  standardized,  and  only 
those  employed  that  have  proved  satisfactory. 

Glassware. — It  is  highly  important  that  all  glassware  should  be 
free  from  clinging  particles  or  traces  of  albumin,  acids,  and  alkalis. 
Pipets  and  dialyzing  cylinders  should  be  washed  in  water,  alcohol, 
ether,  and  finally  in  distilled  water,  and  sterilized  by  dry  heat.  Boiling 
rods  of  solid  glass  (10  cm.  by  0.5  cm.)  should  be  washed  in  alcohol, 
ether,  and  distilled  water,  wrapped  in  bundles  of  six  in  newspaper,  and 
sterilized  by  dry  heat. 

A  very  convenient  dialyzing  cylinder  is  shown  in  the  accompanying 
illustration  (Fig.  76).  This  cylinder  measures  8  by  3  cm.  It  should 
be  plugged  with  cotton  and  sterilized.  When  the  shell  is  loaded  with 
coagulated  placenta  and  serum  and  covered  with  toluol,  it  will  rest  well 
beneath  the  surface  of  the  outside  distilled  water.  The  wide  mouth  of 
the  cylinder  facilitates  all  manipulations  and  the  shell  cannot  upset. 
The  apparatus  is  easily  sterilized,  and  the  cotton  plug  prevents  bac- 
terial contamination  and  undue  evaporation  of  the  contents. 

General  Precautions. — According  to  Abderhalden,  the  work  should 


ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY 


267 


i 


be  conducted  in  a  special  room,  where  there  is  no  dust  or  fumes  of  acids, 

and  where  no  bacteriologic  work  is  in  progress.     This  observer  also 

recommends  that  a  special  incubator  be  used  for  this  work.     If,  however, 

the  working    table    is    scrupulously 

clean  and  the  glassware  is  clean  and 

sterile,  and  if  the  shells  are  handled 

with  sterile  forceps  and  the  dialyzing 

cylinder  is  stoppered  with  a  plug  of 

sterile  cotton,   all  requirements  are 

practically  fulfilled. 

Reagents. — The  presence  of  albu- 
min or  its  split  products  may  be 
detected  by  two  color  reactions:  (1) 
the  biuret  reaction,  and  (2)  the  nin- 
hydrin  reaction.  The  first  is  espe- 
cially delicate  for  uncleaved  albumin, 
and  the  latter  for  peptones  and  amino- 
acids.  The  technic  of  the  biuret  test 
is  described  with  the  technic  of  testing 
shells  for  permeability  to  albumins. 

Ninhydrin.  —  This  is  the  trade 
name  for  triketohydrindenhydrate. 
It  is  a  whitish  yellow,  readily  soluble 
powder,  dispensed  in  brown  glass 
vials  containing  0.1  gram  of  the  drug. 
A  circular  describing  its  method  of 
use  accompanies  each  package.  As 
0.2  c.c.  of  a  1  per  cent,  watery  solu- 
tion is  the  amount  necessary  for  a 
test,  the  contents  of  the  vial  are  dis- 
solved in  10  c.c.  of  distilled  water, 
and  the  vial  rinsed  with  a  portion/ 
of  the  solvent.  This  solution  should 

be  preserved  in  a  brown  bottle  in  a  cold  place,  and  precautions  taken 
to  prevent  infection.  Triketohydrindenhydrate  has  been  described  by 
Ruheman,1  who  gives  its  formula  as  follows: 

C6H/      ^C(OH)2 


FIG.  76. — A  DIALYZING  CYLINDER 
FOR  THE  ABDERHALDEN  FERMENT 
TEST. 

The  shell  contains  placenta! 
tissue  and  fresh  serum;  it  is  sur- 
rounded with  20  c.c.  of  distilled  water 
and  covered  with  toluol.  The  cotton 
plug  prevents  contamination.  The 
cylinder  is  readily  sterilized  in  a  hot- 
air  oven  and  affords  a  simple  and 
efficient  means  for  conducting  the 
test  by  the  dialyzation  method. 


1  Jour.  Chem.  Soc.,  London,  1910,  xcvii,  2025. 


268  FERMENTS   AND   ANTIFERMENTS 

Owing  to  the  fact  that  it  gives  a  blue  color  in  the  presence  of  any 
compound  that  possesses  an  ammo-group  in  the  alpha  position  of  the 
carboxyl  group,  it  is  of  great  value  as  an  aid  in  recognizing  the  products 
of  protein  digestion  (Fig.  77). 

Testing  the  Shell  for  Non-permeability  to  Albumin.—!.  New  shells 
should  be  softened  by  soaking  them  for  half  an  hour  in  sterile  distilled 
water.  A  dozen  or  more  may  be  tested  at  one  time. 

2.  The  albumin  solution  is  prepared  by  placing  5  c.c.  of  the  albu- 
min of  fresh  eggs  in  a  mixing  cylinder,  and  adding  distilled  water  to 
make  100  c.c.     Mix  well.     There  must  be  no  flakes.     Instead,  a  clear, 
hemoglobin-free  serum  which  has  been  dialyzed  against  running  water 
to  remove  dialyzable  substances,  may  be  used  in  doses  of  2.5  c.c.  for 
each  shell. 

3.  Carefully  pipet  5  c.c.  of  the  albumin  solution  into  each  shell. 
Great  care  should  be  exercised  that  none  of  the  solution  contaminates 
the  outside  of  the  shell.    The  preferable  method  is  to  hold  the  shell  with 
a  pair  of  broad-toothed  sterilized  forceps  and  carry  the  pipet  to  the 
bottom,  in  order  that  none  of  the  albumin  should  contaminate  the  upper 
portion  of  the  inside  of  the  shell.    The  pipet  may  easily  touch  the  edge 
of  the  shell  and  thus  contaminate  the  dialysate.    If  in  doubt,  cover  the 
upper  end  of  the  shell  with  the  forceps  and  wash  the  outside  with  running 
water. 

4.  The  loaded  shell  is  now  placed  in  a  sterile  dialyzing  cylinder  con- 
taining 20  c.c.  of  sterile  distilled  water.     Never  load  the  shell  in  this 
cylinder,  for  some  of  the  albumin  may  fall  into  the  distilled  water. 

5.  Cover  the  contents  of  the  shell  and  the  surrounding  distilled  water 
with  a  layer  of  toluol  about  J^  inch  in  depth.     Replace  the  cotton  plug 
in  the  cylinder. 

6.  Incubate  at  37°  C.  for  sixteen  hours. 

7.  Pass  a  sterile  pipet  quickly  through  the  layer  of  toluol  and  remove 
10  c.c.  of  the  dialysate  to  a  clean  sterile  test-tube,  and  test  for  albumin 
by  the  biuret  reaction.     Add  2.5  c.c.  of  a  33  per  cent,  solution  of  sodium 
hydroxid;  shake  gently,  but  remove  the  thumb  from  the  top  of  the  tube. 
The  solution  may  become  slightly  cloudy.     Carefully  overlay  with  1  c.c. 
of  a  0.2  per  cent,  solution  of  copper  sulphate  in  such  manner  that  a  sharp 
line  of  demarcation  separates  the  alkaline  dialysate  from  the  copper 
sulphate  solution.     A  delicate  violet  tint  at  this  line  indicates  that  al- 
bumin is  present  and  that  the  shell  is  useless.     If  one  cannot  see  this 
color  or  is  in  doubt,  it  is  well  to  make  the  ninhydrin  test.     To  do  this 
dialysis  should  be  continued  for  twenty-four  hours;    ninhydrin  reacts 


FIG.    77. — NINHYDEIN   REACTION    (ABDERHALDEN   FERMENT   TEST). 
The  tube  on  the  left  shows  a  positive  reaction  with  the  serum  of  a  pregnant 
woman;  the  tube  on  the  right  is  the  serum  control  and  shows  a  faint  violet  color,  due, 
presumably,  to  the  passage  of  dialyzable  substances  in  this  serum. 


ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY  269 

with  albumin  in  addition  to  peptones  and  amino-acids,  but,  according 
to  Abderhalden,  this  test  is  less  sentitive  than  the  biuret  test. 

8.  All  shells  should  react  negatively, — i.  e.,  they  should  not  permit 
the  passage  of  unchanged  albumin.  If  the  ninhydrin  test  is  used,  the 
tubes  should  be  inspected  one-half  hour  after  boiling,  and  the  contents 
should  be  as  clear  as  water  or  show  but  the  faintest  blue  tint.  If  this  is 
not  the  case,  shells  should  be  discarded  as  being  permeable  to  albumin. 
Those  that  are  satisfactory  in  this  respect  should  be  tested  further  as 
follows : 

Testing  the  Shell  for  Permeability  to  Peptone. — 1.  The  shells  should 
now  be  thoroughly  cleansed,  but  not  with  a  stiff  brush,  washed  in 
running  water,  and  boiled  for  thirty  seconds. 

2.  Prepare  a  1  per  cent,  solution  of  silk  peptone  (Hochst)  in  dis- 
tilled water,  and  carefully  pipet  2.5  c.c.  into  each  shell,  using  every  pre- 
caution against  contaminating  the  upper  portion  on  the  inside,  and 
especially  of  the  outside,  of  the  shell. 

3.  Place  the  loaded  shell  in  a  sterile  dialyzing  cylinder  containing 
20  c.c.  of  sterile  distilled  water,  and  cover  the  contents  of  the  shell  and 
water  with  toluol.     Replace  the  cotton  plug  and  incubate  at  37°  C.  for 
twenty-four  hours. 

4.  Remove  10  c.c.  of  the  dialysate  (avoid  removing  toluol)  to  a 
clean,  sterile,  thin-walled  test-tube,  and  add  0.2  c.c.  of  the  1  per  cent, 
ninhydrin  solution.     Insert  a  sterile  boiling  rod  and  boil  for  exactly  one 
minute. 

5.  The  boiling  process  is  quite  an  important  feature  of  this  test. 
Always  boil  in  precisely  the  same  manner.     A  high  Bunsen  flame  should 
be  used,  and  about  one  minute  after  air-bubbles  first  appear  on  the  sides 
of  the  tube  lively  boiling  commences.     The  flame  should  then  be  turned 
down  and  the  boiling  continued  for  exactly  one  minute. 

6.  Place  the  tube  in  a  rack.     With  a  fresh  sterile  pipet  remove  10 
c.c.  of  dialysate  from  the  next  cylinder  and  test  in  the  same  manner, 
and  repeat  until  the  entire  series  have  been  finished. 

7.  After  half  an  hour  inspect  all  the  tubes;    they  should  show  a 
deep  blue  color;    if  they  do  not  do  so  they  are  impermeable  or  partly 
permeable  to  peptone  and  should  be  discarded.     There  is  usually  a 
difference  in  the  degree  of  color  reaction  among  a  number  of  shells,  as 
their  permeability  varies. 

8.  Those  shells  that  have  withstood  both  tests  are  now  thoroughly 
washed  in  running  water,  boiled  for  thirty  seconds,  placed  in  a  jar 
of  sterile  distilled  water  containing  a  few  drops  of  chloroform,  and 


270  FERMENTS   AND    ANTIFERMENTS 

covered  with  toluol.  From  this  time  on  they  should  not  be  handled 
with  the  fingers,  but  only  with  forceps  that  have  been  sterilized  by 
boiling.  Of  the  entire  number  of  shells,  usually  from  20  to  30  per  cent, 
or  more  are  found  to  be  unsatisfactory. 

Preparation  of  the  Placental  Tissue. — This  is  the  substratum,  and 
should  consist  of  coagulated  placental  protein  free  from  dialyzable  sub- 
stances that  react  with  ninhydrin. 

1.  A  fresh  normal  placenta  should  be  prepared  soon  after  delivery. 
It  is  highly  important  to  wash  it  free  from  all  blood,  Abderhalden  having 
laid  considerable  stress  upon  this  point.     He  explains  that  in  the  blood 
of  all  animals  there  is  always  a  specific  ferment  for  the  red  blood-cor- 
puscles, as  even  the  smallest  hemorrhage  into  the  tissue  calls  forth  a 
protective  ferment.     For  this  reason  all  organs  that  contain  blood  may 
contain  the  substratum  and  ferment,  and  yield  false  positive  reactions. 

2.  The  placenta  should  be  placed  in  warm  water  and  freed  as  far 
as  possible  of  clots.     The  membranes  and  cord  are  removed,  and  the 
placental  tissue  cut  into  pieces  about  the  size  of  a  dime.      These  are 
placed  in  a  sieve  under  running  water,  and  each  piece  squeezed  with  the 
hand.     From  time  to  time  the  entire  mass  is  thoroughly  squeezed  out 
in  a  towel.     Tissues  that  cannot  be  freed  from  clots  should  be  discarded. 
The  tissues  are  now  crushed  in  a  mortar,  connective-tissue  strands  re- 
moved, and  the  washing  continued  until  the  tissue  is  snow  white.     De- 
colorizing substances,  such  as  H202,  should  not  be  used.     If  the  tissue  is 
not  white  and  free  from  blood  it  should  not  be  employed.     Liver,  spleen, 
and  kidney  tissue  cannot  be  made  perfectly  white,  although  all  traces  of 
blood  have  been  removed. 

3.  Place  100  times  as  much  distilled  water  as  there  is  tissue  in  an 
enameled  vessel;  to  each  liter  add  five  drops  of  glacial  acetic  acid  and 
heat  to  boiling,  when  the  tissue  is  added  and  boiled  for  ten  minutes. 

4.  Wash  the  coagulated  tissue  with  distilled  water,  and  boil  again 
without  the  addition  of  acid.     This  should  be  repeated  six  times  in 
succession.     If  an  interruption  occurs,  cover  the  tissue  and  water  with 
a  layer  of  toluol. 

5.  After  the  sixth  boiling  add  a  small  quantity  of  water  to  the  tissues 
— just  sufficient  to  enable  it  to  boil  for  about  five  minutes  without  burn- 
ing, for  the  water  is  now  to  be  tested  with  the  ninhydrin  reaction  and 
it  is  important  that  this  be  as  concentrated  as  possible.     Filter  the  water, 
and  to  5  c.c.  in  a  sterile  test-tube  add  1  c.c.  of  the  ninhydrin  solution. 
Boil  vigorously  for  one  minute.     If  there  is  the  slightest  discoloration 
within  half  an  hour,  the  tissues  must  be  boiled  again,  but  with  only  five 


ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY  271 

volumes  of  water  and  no  longer  than  five  minutes  each  time.  These 
boilings  should  be  repeated  as  often  as  is  necessary  until  the  ninhydrin 
reaction  remains  water  clear  for  at  least  one-half  hour. 

6.  The  tissues  are  again  gone  over  with  a  sterile  forceps,  and  a  search 
made  for  brown  masses  resembling  blood-clots.     These  are  to  be  dis- 
carded. 

7.  The  tissue  is  now  preserved  hi  a  sterile  jar  containing  sufficient 
sterile  water  and  chloroform  and  covered  with  toluol.     All  tissue  should 
be  handled  with  sterile  forceps,  and  when  once  removed  from  the  jar, 
they  should  never  be  returned.     The  whole  operation  requires  several 
hours  and  it  should  be  conducted  without  interruption.     If  the  process 
is  interrupted,  the  tissue  should  be  covered  with  a  layer  of  toluol. 

8.  It  is  well  to  try  out  the  tissue  with  a  known  serum  of  pregnancy 
to  make  certain  that  it  is  a  suitable  substratum. 

9.  Only  normal  placenta  should  be  used,  as  in  certain  instances  a 
normal  organ  may  be  satisfactory,  whereas  a  diseased  organ  would  be 
unsuitable. 

10.  Animal  placenta  may  be  substituted  for  human  placenta  and 
vice  versa,   but  Abderhalden  cautions  against  this  substitution  until 
further  work  has  been  done. 

The  Blood-serum. — The  serum  to  be  tested  must  fulfil  three  condi- 
tions: 

(1)  It  must  contain  the  smallest  amount  of  dialyzable  substances 
that  would  react  with  ninhydrin.     Blood  is  best  drawn  in  the  morning 
before  breakfast.     In  all  diseases  accompanied  by  marked  protein  dis- 
integration, such  as  cancer,  the  blood-serum  may  contain  large  amounts 
of  dialyzable  substances. 

(2)  It  must  be  absolutely  free  from  hemoglobin  and  clear. 

(3)  It  must  be  free  from  cells.      Even  an  apparently  clear  serum  may 
contain  millions  of  erythrocytes. 

1.  From  10  to  20  c.c.  of  blood  are  withdrawn  from  a  vein  at  the 
elbow  with  a  dry  sterile  needle  into  a  sterile  centrifuge  tube.     This  is 
placed  aside  at  room  temperature  for  several  hours,  when  sufficient 
serum  has  usually  separated  out;  if  this  has  not  occurred,  centrifuge  for 
several  minutes.     The  serum  is  removed  to  a  second  sterile  centrifuge 
tube,  and  centrifuged  at  high  speed  for  several  minutes  until  all  cor- 
puscles have  been  precipitated  to  the  bottom  of  the  tube. 

2.  The  serum  should  be  used  within  twelve  hours  after  the  blood  has 
been  withdrawn.     Abderhalden  claims  that  heating  a  serum  to  60°  C. 
robs  it  of  its  digesting  powers.     Pearce  and  Williams  have  found  that 


272  FERMENTS    AND    ANTIFERMENTS 

inactivation  considerably  weakens  the  reaction,  but  does  not  abolish  it 
altogether. 

3.  Specimens  of  blood  sent  through  the  mails  are  really  unsatisfac- 
tory, for  even  if  they  are  delivered  within  twelve  hours  after  bleeding, 
the  amount  of  handling  has  usually  resulted  in  the  breaking  up  of  a 
number  of  corpuscles  and  the  tinging  of  the,  serum  with  hemoglobin. 

The  Test. — 1.  Absolute  cleanliness  should  be  employed.  The  glass- 
ware should  be  sterile  and  dry,  and  everything  should  be  in  readiness. 
The  technic  should  be  aseptic  and  thoroughly  understood. 

2.  Remove  a  sufficient  amount  of  the  prepared  placenta  for  the  work 
at  hand  with  sterile  forceps  and  wash  in  a  dish  of  sterile  distilled  water 
to  remove  toluol  and  chloroform.    Boil  with  4  or  5  volumes  of  sterile 
distilled  water  for  two  minutes  and  test  the  water  with  ninhydrin. 
If  positive,  the  tissue  must  be  boiled  as  described  above  until  free  of 
ninhydrin  reacting  substances.    Place  on  sterile  filter-paper,  and  squeeze 
to  remove  any  excess  of  water.    Weigh  and  place  0.5  gram  in  each  of 
two  shells  (one  for  a  control). 

3.  Holding  each  shell  with  a  second  pair  of  boiled  forceps,  pipet  1.5 
c.c.  of  the  patient's  serum  into  one  shell  containing  placenta,  and  the 
same  amount  into  a  third  shell  which  is  to  serve  as  a  control  on  the 
serum.     Place  1.5  c.c.  of  sterile  distilled  water  in  the  placenta!  tissue 
control  shell. 

4.  Unless  one  is  absolutely  sure  that  neither  the  tissue  nor  the  serum 
has  touched  the  outside  of  the  shells,  they  should  be  held  shut  with 
sterile  forceps  and  washed  with  sterile  distilled  water. 

5.  Each  of  the  three  shells  is  now  placed  in  cylinders  containing 
20  c.c.  of  sterile  distilled  water.     Under  no  circumstances  are  the  shells 
to  be  loaded  while  they  are  in  the  dialyzing  cylinders. 

6.  The  contents  of  each  shell  and  the  water  surrounding  them  are 
covered  with  a  layer  of  toluol  about  J4  inch  in  depth,  and  the  cylinders 
plugged  with  cotton  to  prevent  evaporation  and  contamination.     The 
shell  should  be  at  least  M  to  J/2  mch  above  the  level  of  the  outside  fluids, 
and  due  care  must  be  exercised  in  carrying  the  cylinder  back  and  forth 
from  the  incubator  that  the  contents  of  the  shell  and  the  surrounding 
water  do  not  become  mixed. 

7.  If  it  is  at  all  possible,  it  is  well  to  set  up  two  more  shells  as  controls, 
each  containing  placenta  and  normal  serum  and  the  serum  of  pregnancy 
respectively. 

8.  All  the  cylinders  are  incubated  at  37°  C.  for  twenty-four  hours. 
Ten  c.c.  of  the  dialysate  are  then  removed  from  each  tube  with  a  sepa- 


ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY  273 

rate  sterile  pipet  and  placed  in  sterile  test-tubes  of  the  same  size  and 
boiled  with  0.2  c.c.  of  the  1  per  cent,  ninhydrin  solution  for  exactly  one 
minute.  After  standing  for  half  an  hour,  the  readings  are  made. 

Bronfenbrenner's  Modification. — Bronfenbrenner1  has  resolved  the 
technic  of  the  test  into  two  distinct  phases :  The  first  comprises  a  sensi- 
tization  of  the  substrat  by  the  specific  elements  of  the  immune  serum 
at  a  low  temperature,  with  the  resultant  absorption  of  antiferment; 
the  second  is  the  autodigestion  of  the  serum  at  37°  C.  In  conducting 
the  test  0.5  gram  of  prepared  placenta  is  mixed  in  a  sterile  tube  with  1.5 
c.c.  serum  and  held  in  the  cold  for  sixteen  hours.  The  clear  serum  is  then 
secured  by  thorough  centrifuging  and  placed  in  a  dialyzing  thimble  in 
distilled  water  as  described  above,  incubated  for  eighteen  to  twenty-four 
hours,  and  the  dialysate  tested  with  ninhydrin.  As  a  further  test  the 
substrat  may  be  washed  twice  with  sterile  distilled  water  by  centrifuging 
to  remove  all  traces  of  serum  and  placed  in  a  dialyzing  thimble  with 
1.5  c.c.  fresh  male  guinea-pig  serum.  The  test  is  then  conducted  in  the 
same  manner  as  the  regular  Abderhalden  technic  and  the  usual  controls 
are  included.  It  would  appear  that  this  modification  has  increased 
the  specificity  of  the  test. 

Reading  the  Reaction. — The  dialysate  of  the  serum  alone  should  be 
clear  as  water  or  show  but  the  faintest  blue  tinge.  The  dialysate  of  the 
placenta  alone  should  be  clear;  the  dialysate  of  the  patient's  serum 
plus  that  of  the  placenta  may  show  a  deep  violet-blue  color  when  the 
reaction  is  strongly  positive,  or  a  fainter  blue  when  it  is  weakly  positive. 
If  this  dialysate  is  water  clear  or  has  a  faint  blue  color,  comparable  to 
the  controls,  the  result  is  negative.  If  there  is  any  doubt,  the  test 
should  be  repeated.  The  negative  control  should  be  water  clear  or  have 
a  faint  tinge  comparable  to  its  control.  The  positive  control  should 
show  a  deep  violet-blue  color. 

I  generally  control  the  result  given  by  the  shell  containing  tissue  and 
patient's  serum  by  cleansing  it  thoroughly,  boiling  for  a  minute,  and 
testing  it  with  egg-albumen  solution  or  a  serum,  in  case  the  reaction 
was  positive,  to  make  sure  that  the  shell  has  not  allowed  the  passage  of 
serum,  or  with  peptone  solution,  in  case  the  reaction  was  negative,  to 
make  sure  that  it  was  not  thick  enough  to  block  the  passage  of  peptones 
and  amino-acids.  This  procedure  delays  the  report  on  a  serum  for  an- 
other twenty-four  hours,  but  the  greater  accuracy  obtained  warrants  the 
delay. 

Readings  should  never  be  made  by  artificial  light.     Tubes  should  be 

1  Jour.  Lab.  and  Clin.  Med.,  1915,  1,  79. 
18 


274  FERMENTS    AND    ANTIFERMENTS 

held  against  a  white  background  the  better  to  appreciate  the  color 
changes. 

A  pinkish  or  brownish  yellow  discoloration  has  nothing  to  do  with 
the  ninhydrin  reaction. 

Sources  of  Error  in  the  Dialyzation  Method. — There  are  many 
sources  of  error,  and  until  the  technic  has  been  improved  sufficiently  to 
eliminate  these,  Abderhalden's  directions  should  be  followed  minutely. 

1.  The  shells  may  become  spoiled  in  time.     They  should  not  be 
cleansed  with  rough  brushes   or   boiled  too   long.     They  should   be 
cleansed  at  once  after  using,  and  tested  every  four  weeks.     If  a  wrong 
diagnosis  results,  the  shell  should  be  retested  at  once. 

2.  The  placental  tissue  is  an  important  source  of  error,  due  to  the 
fact  that  it  contains  blood. 

3.  The  serum  should  be  fresh  and  free  from  hemoglobin  and  cor- 
puscles. 

4.  The  controls  on  placenta  alone  and  each  serum  alone  are  abso- 
lutely necessary,  as  both  may  contain  various  substances  capable  of 
reacting  with  ninhydrin  and  thus  yielding  false  positive  reactions. 

5.  The  water  used  should  be  distilled  and  sterile.     The  glassware 
should  be  chemically  clean  and  sterile,  and  the  laboratory  free  from  the 
fumes  of  acids  and  alkalis.     It  is  very  important  that  absolutely  the 
same  conditions  should  exist  for  the  control  tests  as  for  the  main  test 
itself. 

THE  OPTICAL  METHOD 

In  the  dialyzation  method,  we  establish  the  transformation  of  a 
colloid  into  a  diffusible  crystalloid;  in  the  optical  method  we  start, 
for  purely  technical  reasons,  not  with  the  whole  protein  molecule,  but 
with  a  peptone  prepared  of  placental  protein.  The  unsplit  protein  it- 
self cannot  be  used,  as  this  will  interfere  with  the  determination  of  the 
rotation  of  the  mixture  of  substratum  plus  serum.  Further,  in  such 
mixtures  precipitation  may  occur  and  render  the  readings  difficult. 
Instead,  we  transform  the  protein  into  peptone,  and  observe  the  final 
changes  in  the  tube  of  the  polariscope. 

Placental  Peptone. — This  requires  considerable  care  in  its  prepara- 
tion. Placental  peptone  may  be  purchased  of  the  Hochst  Farbwerke, 
and  is  expensive.  Each  specimen  should  be  tested  and  its  rotation  de- 
termined, as  otherwise  uncertain  and  unreliable  results  may  be  secured. 
According  to  Abderhalden,  a  peptone  may  be  prepared  as  follows: 


ABDERHALDEN'S  SERODIAGNOSIS  OF  PREGNANCY  275 

The  tissues  are  first  cut  into  small  pieces  and  thoroughly  washed  until  they  are 
white,  as  in  the  preparation  of  tissues  for  the  dialyzation  method. 

The  tissue  is  freed  of  any  excess  of  water  by  pressing  it  through  several  layers  of 
filter-paper,  and  the  process  of  hydrolysis  is  begun.  If  a  larger  quantity  of  the  same 
tissue  is  to  be  collected,  the  pieces  are  prepared  as  they  are  secured  by  washing  them 
free  from  blood,  boiling  in  water  for  ten  minutes,  and  preserving  in  a  stock  jar  con- 
taining sterile  water  and  chloroform,  and  covered  with  toluol.  The  tissues  are  boiled 
in  order  to  destroy  the  cell  ferments  and  to  prevent  autolysis. 

The  tissues  are  now  weighed,  placed  in  a  large  flask,  and  treated  with  three 
volumes  of  cold  70  per  cent,  sulphuric  acid.  The  flask  is  well  shaken  and  carefully 
stoppered,  and  placed  aside  at  room  temperature  (not  higher  than  20°  C.)  until  the 
tissues  have  gone  into  solution.  The  flask  is  shaken  occasionally.  Solution  is 
usually  completed  within  three  days.  The  flask  is  now  placed  in  iced  water  and 
treated  with  10  volumes  of  distilled  water  added  slowly  so  that  the  temperature 
does  not  rise  above  20°  C. 

The  sulphuric  acid  is  now  precipitated  with  pure  crystallized  barium  hydroxid 
until  the  solution  gives  no  precipitate  with  either  the  hydroxid  or  sulphuric  acid. 
A  precipitate  of  a  barium  salt  of  peptones  may  appear  in  spite  of  the  fact  that  no 
sulphuric  acid  is  present.  This  precipitate  is  soluble  in  nitric  acid,  whereas  barium 
sulphate  is  not  soluble.  The  neutralization  point  is  controlled  with  litmus  paper. 
Small  portions  are  then  filtered  through  filter-paper  and  tested,  first  with  barium 
hydroxid  and  then  with  sulphuric  acid.  If  a  turbidity  develops  on  testing  with  the 
barium  hydroxid,  nitric  acid  is  added  and  the  whole  gently  warmed.  If  the  pre- 
cipitate persists,  it  is  evident  that  more  barium  sulphate  should  be  added.  The 
final  neutralization  is  effected  with  dilute  sulphuric  acid  and  barium  hydroxid. 
When  the  solution  is  free  from  sulphuric  acid,  the  precipitate  of  barium  sulphate  is 
secured  by  filtering  through  filter-paper  or  by  centrifugalization.  It  is  then  worked 
up  in  a  mortar  with  distilled  water  and  again  filtered.  It  is  well  to  repeat  this  wash- 
ing once  more. 

The  peptone  solution  is  now  concentrated  in  a  special  apparatus  that  permits 
evaporation  of  the  solution  under  diminished  pressure  and  at  40°  C.  A  special  drop 
funnel  delivers  the  peptone  solution  drop  by  drop  and  thus  prevents  foaming. 

The  yellowish,  syrupy  residue  that  remains  is  covered  with  100  volumes  of 
methyl  alcohol  and  boiled.  The  boiling  hot  solution  is  then  filtered  through  five 
thicknesses  of  filter-paper  into  five  volumes  of  cold  ethyl  alcohol,  which  is  kept  in 
ice  water.  Precipitation  may  be  accomplished  by  the  addition  of  ether.  Just  as 
soon  as  the  precipitate  has  formed  it  is  filtered  out,  preferably  through  a  porcelain 
filter.  The  filter  is  then  placed  in  a  vacuum  desiccator,  and  after  one  or  two  days 
the  peptone  is  dry  and  easily  removed  and  weighed. 

A  10  per  cent,  solution  in  0.9  per  cent,  salt  solution  is  prepared,  and  the  rotation 
determined.  If  the  rotation  is  more  than  1°,  the  solution  is  diluted  until  the  rota- 
tion is  0.75°. 

Testing  the  Peptone. — One  cubic  centimeter  of  fresh,  clear,  hemo- 
globin- and  corpuscle-free  serum  from  a  man  and  an  equal  amount  of  a 
5  to  10  per  cent,  solution  of  the  peptone  are  placed  in  a  sterile  polaris- 
cope  tube,  warmed  to  37°  C.,  and  the  rotation  read.  If  marked  changes 
occur,  the  peptone  is  not  free  from  sulphuric  acid  or  barium  hydroxid. 
With  the  serum  of  pregnancy,  readings  should  be  taken  each  hour  for 
six  hours,  and  then  at  intervals  of  from  thirty-six  to  forty-eight  hours. 


276  FERMENTS   AND   ANTIFERMENTS 

It  is  well  to  test  a  number  of  pregnancy  serums  until  a  normal  curve 
can  be  charted. 

Peptones  of  other  tissues  and  bacteria  may  also  be  prepared. 

The  Polariscope. — A  perfect  and  delicate  instrument  is  necessary, 
that  of  Schmidt  and  Hansch,  Berlin,  being  recommended  by  Abder- 
halden.  The  instrument  must  be  delicate  enough  to  record  differences 
in  rotation  of  0.01 p,  and  be  furnished  with  an  electric  incubator  attach- 
ment for  keeping  the  tube  at  a  constant  temperature  of  37°  C.  The 
tubes  may,  however,  be  kept  in  a  bacteriological  incubator,  removed, 
quickly  read,  and  then  returned. 

The  Test. — One  cubic  centimeter  of  fresh  and  absolutely  hemoglo- 
bin- and  corpuscle-free  serum  is  placed  in  the  polarization  tube  with 
1  c.c.  of  a  10  per  cent,  solution  of  standardized  placental  peptone;  suf- 
ficient sterile  saline  solution  is  added  to  fill  the  tube.  The  tube  is  then 
placed  in  an  incubator  at  37°  C.  for  an  hour,  and  a  reading  made.  An- 
other reading  is  taken  an  hour  later,  and  the  two  readings  should  not 
show  more  than  a  minute  difference  in  rotation.  Another  reading  is 
made  at  the  end  of  six  hours,  and  others  at  intervals  during  the  next 
thirty-six  or  forty-eight  hours. 

Reading  the  Reaction. — In  serums  of  pregnancy  cleavage  is  usually 
apparent  at  the  end  of  six  hours,  and  rotation  may  amount  to  0.05°  to 
0.2°  in  thirty-six  hours.  With  non-pregnant  serums  the  rotation  is 
seldom  more  than  0.03°. 

Considerable  experience  is  required  in  making  these  readings,  and 
the  novice  should  practise  well  before  conducting  diagnostic  tests. 

Individual  readings  by  different  persons  may  vary  0.02°,  and  in 
order  to  secure  absolute  certainty  Abderhalden  gives  0.04°  as  the  limit 
for  error. 

A  large  margin  for  error  will  result  if  the  primary  reading  is  taken 
when  the  tube  is  cold,  as  it  is  immediately  after  being  filled.  Only  that 
reading  taken  after  the  tube  has  been  in  the  incubator  for  at  least  one  or 
two  hours  is  to  be  taken  as  the  guide  for  determining  the  amount  of 
digestion  according  to  the  degree  of  -rotation. 

The  greatest  source  of  error  lies  in  the  observer  himself.  One  must 
be  trained  to  make  the  readings  in  about  thirty  seconds;  the  eye  soon 
grows  tired,  and  the  readings  are  then  unreliable. 

PRACTICAL  VALUE  OF  ABDERHALDEN'S  PREGNANCY  TEST 
1.  It  may  be  definitely  stated  that  this  test  has  failed  to  establish 
itself  as  possessing  value  in  the  diagnosis  of  pregnancy.     When  per- 


SERO-ENZYMES    IN   DISEASE  277 

formed  by  a  competent  serologist  following  all  the  prescribed  precautions, 
satisfactory  results  may  be  obtained  in  some  cases,  but  errors  beyond 
the  immediate  control  of  the  serologist  are  prone  to  be  present  and 
greatly  limit  the  practical  value  of  the  test. 

2.  As  the  test  is  more  likely  to  yield  falsely  positive  than  negative 
results,  a  negative  reaction  is  of  some  value  in  excluding  pregnancy, 
and  particularly  in  those  occasional  cases  presenting  tumors  in  the 
lower  abdomen  which  may  be  due  to  advanced  pregnancy  or  a  neoplasm. 

3.  The  reaction  may  appear  in  the  middle  of  the  second  month,  and 
disappear  in  from  ten  to  fifteen  days  after  pregnancy  has  been  interrupted, 
regardless  of  whether  the  fetus  is  born  before,  at,  or  after  the  normal 
period  of  gestation.    This  test  has  generally  failed,  however,  to  reliably 
detect  pregnancy  at  a  time  when  such  a  test  is  mostly  needed,  namely, 
in  the  early  stages  and  when  the  clinical  diagnosis  is  doubtful. 

4.  As  it  stands  at  present,  the  Abderhalden  test  has  only  a  scientific 
interest,  but  is  worthy  of  further  investigation;  it  has  greatly  stimulated 
the  study  of  the  fermentative  activities  of  the  body  fluids  and  particu- 
larly of  the  blood. 

SERO-ENZYMES  IN  DISEASE 

Cancer. — Freund  and  Abderhalden1  claim  to  have  found  ferments 
in  the  serum  of  cancer  that  will  digest  coagulated  cancer  protein  in  the 
same  manner  as  the  ferments  in  pregnancy  digest  placental  protein. 
Frank  and  Heiman2  reported  positive  results  in  53  of  54  cases  of  cancer; 
Markins  and  Munze,3  Epstein,4  Gambaroff,5  Erpicum,6  Brockman,7 
Lampe,8  Lowy,9  Ball,10  and  others  have  reported  highly  favorable  results. 
Frankle11  and  Lindig12  have  found  the  reactions  generally  non-specific 
in  character. 

Either  the  dialyzation  or  the  optical  method  may  be  used  in  conduct- 
ing these  tests,  precisely  the  same  technic  being  followed  as  in  making 
the  pregnancy  tests,  except  that  several  different  cancer  tissues  should  be 

I  Munch,  med.  Wochenschr.,  1913,  14,  763. 
2Berl.  klin.  Wochenschr.,  1913,  1,  No.  14. 

3  Berl.  klin.  Wochenschr.,  1913,  1,  No.  17. 
4Wien.  klin.  Wochenschr.,  1913,  xxvi,  No.  17. 

5  Berl.  klin.  Wochenschr.,  1913,  1,  No.  17. 

6  Bull,  de  1'Acad.  Roy.  de  Belg.,  1913,  xxvii,  624. 

7  Lancet,  London,  November  15,  1913. 

8  Munch,  med.  Wochenschr.,  1914,  Ixi,  No.  9. 

9  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  437. 
10  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  599. 

II  Deutsch.  med.  Wochenschr.,  xl,  No.  12. 

12  Munch,  med.  Wochenschr.,  1913,  60,  288. 


278 


FERMENTS   AND    ANTIFERMENTS 


used  with  each  serum.  It  is  well  to  make  the  experiment  with  a  number 
of  cancer  tissues  taken  from  various  parts,  also  with  sarcoma  tissue  and 
that  of  various  benign  tumors. 

Mental  Diseases. — Fauser1  has  studied  the  serums  of  88  cases  of 
dementia  pra?cox  and  other  mental  diseases  with  various  antigens  com- 
posed of  the  ductless  glands,  testicles,  ovaries,  etc.,  and  attained  in- 
teresting results,  tending  to  show  that  in  many  of  these  brain  affections 
there  may  be  associated  lesions  in  other  organs,  and  that  the  symptoms 
may  be  due  to  perverted  functions  of  certain  ductless  glands.  Munzer,2 
Bundschue  and  Roener,3  and  Fisher4  have  also  found  in  the  serums  of 
mental  and  nervous  diseases  ferments  for  the  protein  of  the  ductless  and 
generative  glands,  tending  to  show  that  lesions  of  these  organs  may  be 
operative  in  the  symptomatology  of  these  conditions. 

Syphilis. — Baeslack5  has  reported  having  had  exceptionally  good 
results  with  the  serums  of  syphilitics  and  a  substratum  composed  of 
coagulated  syphilitic  lesions  of  rabbit's  testicle.  Using  the  dialyzation 
method,  he  found  the  sero-enzyme  test  more  constant  and  earlier  than 
the  Wassermann  reaction. 

Tuberculosis  and  Acute  Infections. — Abderhalden  and  Andryewsky6 
have  suggested  the  use  of  the  dialyzation  or  the  optic  method  in  the 
diagnosis  of  acute  infections.  The  peptone  may  either  be  prepared  of 
the  bacilli,  or  the  boiled  organisms  used  in  the  dialyzing  shell.  In  pre- 
paring a  bacterial  substratum,  the  material  must  be  carefully  centri- 
fuged  in  order  to  facilitate  washing.  The  tubercle  bacilli  are  degreased 
by  extraction  in  fat  solvents.  According  to  Abderhalden,  the  presence 
of  ferments  in  acute  infections  indicates  that  the  animal  is  defending 
itself.  Abderhalden  and  Andryewsky  found  ferments  present  m  the 
serum  of  cattle  receiving  injections  of  suspensions  of  dead  tubercle 
bacilli  and  in  experimental  infections,  and  suggest  that  the  test  may 
prove  efficacious  in  testing  cattle.  This  work  should  receive  further 
study  in  human  infections.  Smith,7  employing  Bronfenbrenner's  modi- 
fication of  the  Abderhalden  test,  has  reported  highly  specific  results 
in  the  differentiation  of  various  bacteria  with  rabbit  immune  sera. 

1  Deutsch.  med.  Wochenschr.,  1913,  xxxix,  No.  7. 

2  Berl.  klin.  Wochenschr.,  1913,  1,  No.  5. 

3  Deutsch.  med.  Wochenschr.,  1913,  No.  42,  2069. 

4  Deutsch.  med.  Wochenschr.,  1913,  No.  44,  2138. 

6  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1002;  ibid.,  Ixiii,  559. 

6  Munch,  med.  Wochenschr.,  1913,  Ixi,  1641. 

7  Jour.  Infect.  Dis.,  1916,  18,  14. 


CHAPTER  XVI 
AGGLUTININS 

As  previously  stated,  given  any  infection,'  several  antibodies  of 
different  properties  may  be  produced.  If  the  infecting  microorganism 
produces  characteristically  an  exogenous  toxin,  as,  for  example,  that 
produced  by  the  diphtheria  bacillus,  an  antitoxin  is  produced  as  the  most 
prominent  of  several  antibodies.  With  other  pathogenic  bacteria  that 
produce  mainly  an  endogenous  toxin  various  antibodies  are  formed,  and 
one  or  more  may  play  a  prominent  role  in  protecting  the  host,  such  as 
opsonins,  agglutinins,  precipitins,  bacteriolysins,  etc. 

If  typhoid  immune  serum  from  an  immunized  animal  or  a  patient 
suffering  from  typhoid  fever  is  added  to  an  emulsion  of  typhoid  bacilli 
in  a  test-tube  and  the  mixture  placed  in  an  incubator,  the  following 
phenomenon  will  be  observed:  the  bacteria,  which  previously  formed  a 
uniform  emulsion,  clump  together  into  little  masses,  settle  at  the  sides 
of 'the  test-tube,  and  gradually  fall  to  the  bottom,  the  fluid  becoming 
almost  clear.  In  a  control  test  to  which  no  active  serum  is  added,  the 
fluid  remains  uniformly  cloudy.  If  the  reaction  is  observed  microscopic- 
ally in  a  hanging  drop,  it  is  noted  that  with  the  addition  of  the  serum  the 
bacilli  move  nearer  and  nearer  one  another,  this  process  being  followed 
by  a  gradual  loss  in  motility  and  the  formation  of  clumps.  The  sub- 
stance in  the  serum  causing  this  phenomenon  is  called  agglutinin,  and 
the  reaction  is  known  as  agglutination. 

Definition. — Agglutinins  are  antibodies  that  possess  the  power  of  caus- 
ing bacteria,  red  blood-corpuscles,  and  some  protozoa  (trypanosomes)  sus- 
pended in  a  fluid  to  adhere  and  form  clumps. 

Historic. — Although  Metchnikoff,  Charrin,  and  Roger  had  noticed 
peculiarities  in  the  growth  of  Bacillus  pyocyaneus  when  cultivated  in 
immune  serum  which  we  now  believe  were  due  to  agglutinins,  Gruber 
and  Durham  and  Bordet  (1894-1896)  were  the  first  to  recognize  that 
the  agglutination  reaction  was  a  separate  function  of  immune  serum. 
While  investigating  the  Pfeiffer  phenomenon  of  bacteriolysis  with  Bacil- 
lus coli  and  the  cholera  vibrio,  these  investigators  found  that  if  the 
respective  immune  serums  were  added  to  bouillon  cultures  of  these  two 

279 


280 


AGGLUTININS 


species,  the  cultures  would  lose  their  turbidity,  flake-like  clumps  would 
form  and  sink  to  the  bottom  of  the  tube,  and  the  supernatant  fluid 
would  become  clear.  Gruber  at  the  same  time  called  attention  to  the 
fact  that  agglutinins  were  not  absolutely  specific  for  their  own  antigen, 
but  would  agglutinate,  to  a  lesser  extent,  closely  allied  species  of  bacteria. 
In  1896  Pfaundler  drew  attention  to  a  peculiar  phenomenon  ob- 
served when  bacteria  were  grown  in  an  immune  serum.  Long  and  more 
or  less  interlaced  threads  of  bacteria  developed,  which  were  regarded  as 
due  to  agglutinins.  At  that  time  considerable  emphasis  was  laid  upon 
the  importance  of  Pfaundkr's  reaction,  but  at  present  the  ordinary 


J  YAp* 

%^/^s 

^     If  /; 


FIG.  78. — THEORETIC  FORMATION  OF  AGGLUTININS. 

agglutination  tests  have  superseded  this  reaction  as  a  practical  diag- 
nostic procedure. 

In  1896  Widal  and  Griinbaum  first  turned  these  facts  to  practical 
use  in  the  diagnosis  of  typhoid  fever.  These  investigators  found  that 
the  serum  of  patients  suffering  from  typhoid  fever  acquires  a  high  agglu- 
tinating power  for  Bacillus  typhosus,  and  since  this  phenomenon  gen- 
erally manifests  itself  comparatively  early  in  the  disease,  its  recognition 
has  considerable  diagnostic  importance.  It  is  purely  accidental  that 
we  speak  of  the  "Widal  reaction"  in  typhoid  fever,  rather  than  of  the 
"Griinbaum  reaction,"  for  the  latter  observer  conducted  similar  studies 


FORMATION   OF  AGGLUTININS  281 

independent  of  Widal,  but,  owing  to  a  lack  of  patients,  Widal  preceded 
him  in  the  publication  of  a  more  extensive  work. 

At  the  present  time  this  diagnostic  reaction  is  known  as  the  Gruber- 
Widal  reaction.  It  has  proved  of  great  value  to  a  large  number  of  dif- 
ferent investigators,  not  only  in  making  the  serum  diagnosis  of  typhoid 
fever,  but  in  other  infections  as  well. 

Normal  and  Immune  Agglutinins.  —  Normal  serums  are  frequently 
capable  of  agglutinating  bacteria,  such  as  the  typhoid,  colon,  pyocy- 
aneus,  and  dysentery  bacilli.  In  some  cases  the  typhoid  bacillus  may  be 
agglutinated  in  dilutions  as  high  as  1  :  30,  a  point  of  practical  impor- 
tance in  the  clinical  use  of  the  test.  When  a  normal  serum  is  found  to 
have  a  high  agglutinating  power,  it  is  probable  that 
a  previous  infection  by  the  microorganism  has  oc- 
curred. Since  the  serum  of  a  new-born  child  is 
largely  devoid  of  agglutinins  that  are  found  in  later 
life,  the  so-called  normal  agglutinins  may,  after  all, 
be  acquired  properties. 

The  term  immune  agglutinin  is  applied  to  the 

RETIC        STRUO 
agglutmatmg  substance  in  a  serum  developed  as  the         TURE  OF  AGGLU- 

result  of   infection  or  of  systematic  immunization         SAJTCNOTO'   AG~ 
with  the  microorganism.  1,   Agglutinin: 


Formation  of  Agglutinins.—  According  to  Ehr-  ap°P 


unon 

lich's  side-chain  theory,  agglutinins  are  antibodies  of  with  antigen;  4,  the 

i       i      /TT      .fOA      r™  agglutinophore     or 

the  second  order  (Fig.  78).   They  resemble  antitoxins  zymophore  group. 

or  receptors  of  the  first  order  in  possessing  an  affinity-  2j  Agglutinoid. 

J  Same  structure  as 

bearing  or  haptophore  group  that  unites  with  the  agglutinin,    except 


antigen,  but  they  differ  from  them  in  having  also  a     phore^r  zymophore 
functional  or  agglutinophore  group  that  agglutinates     grouP  is  lost- 
the  antigen  when  this  union  has  occurred  (Fig.  79). 

Agglutinins  that  have  lost  their  zymophore  or  agglutinophore  group 
through  the  action  of  heat,  age,  acids,  etc.,  but  that  still  possess  their 
haptophore  group,  are  called  agglutinoids,  just  as  toxins  that  have  lost 
their  toxophore  group  are  called  toxoids.  Such  agglutinoids,  then,  may 
still  combine  with  the  bacteria  or  blood-cells  without  being  able,  how- 
ever, to  produce  agglutination  (Fig.  80). 

It  is  found,  at  times,  that  even  a  fresh  serum,  when  concentrated, 
will  cause  less  agglutination  than  when  it  is  diluted.  This  is  ascribed  to 
the  presence  of  agglutinoids,  which  have  a  stronger  affinity  for  agglutin- 
ogen  than  has  the  agglutinin.  When  producing  a  reaction  of  this  char- 
acter they  are  called  pro-aqglutinoids.  When  the  serum  is  diluted,  the 


282 


AGGLUTININS 


pro-agglutinoids  become  less  concentrated,  and  finally,  when  they  are 
diluted  as  to  have  no  influence  on  the  reaction,  the  agglutinins  are  still 
present  in  sufficient  quantity  to  bring  about  agglutination.  As  a  practi- 
cal fact,  in  agglutination  reactions  the  action  of  pro-agglutinoids  is  of 

much  importance,  for  the  inexperi- 
enced may  be  misled  by  the  absence 
of  or  by  poor  agglutination  in  lower 
dilutions  to  neglect  the  use  of  higher 
dilutions  (see  Fig.  86). 

The  substance  in  bacteria  or 
other  cells  that  produces  agglutinin 
is  called  agglutinogen.  It  appears 
to  be  formed  in  the  cell,  and  in 
some  cases  may  be  excreted  into 
the  surrounding  medium.  Certainly 
when  bacteria  die  and  become  dis- 
integrated, agglutinogen  is  liberated 
and  the  filtrates  (entirely  free  from 
bacterial  cells),  when  injected  into 
animals,  will  cause  the  formation  of 
agglutinins. 

Agglutinogen  must  be  con- 
sidered as  having  a  simple  hapto- 
phorous  group,  through  which  it 
may  unite  with  the  receptors  of  the 
tissue-cells.  This  haptophore 
comes  into  play  again  in  the  union 
between  agglutinogen  and  agglu- 
tinin, which  precedes  agglutination. 
It  is  a  passive  body,  similar  to 

the  haptophore  of  antitoxin,  and  has  no  other  function  than  that  of 
uniting  either  with  cell  or  with  agglutinin. 

Origin  of  Agglutinins. — The  investigations  that  have  been  carried 
out  for  the  purpose  of  determining  the  site  of  formation  of  agglutinins 
have  not  thus  far  yielded  conclusive  results.  The  lymphoid  tissues 
appear  especially  concerned,  agglutinins  being  found  early  in  the  bone- 
marrow  and  the  spleen  (Pfeiffer  and  Marx).  Metchnikoff  believes  that 
agglutinins  may  be  derived  from  leukocytes  and  endothelial  cells.  It  is 
more  probable,  however,  that  the  formation  is  general,  and  is  the  result 
of  wide-spread  cellular  activity. 


FIG.  80. — A  DIAGRAMMATIC  ILLUSTRA- 
TION OF  THE  ACTION  OF  AGGLUTI- 
NINS AND  AGGLUTINOIDS. 

In  the  first  tube  (left)  most  of  the 
bacilli  (B)  have  been  agglutinated  and 
massed  in  the  bottom  of  the  tube  by 
the  agglutinins  (a). 

In  the  second  tube  (right)  the 
bacilli  (B)  are  in  combination  with  the 
agglutinoids  (A),  but  agglutination 
does  not  occur  because  the  agglutino- 
phore  groups  are  lost.  A  few  bacilli 
have  been  agglutinated  by  the  agglu- 
tinins (a). 


ACID    AGGLUTINATION  283 

In  accordance  with  the  side-chain  theory,  the  ability  of  an  animal  to 
form  agglutinins  for  a  certain  microorganism  would  depend  on  its  pos- 
session of  receptors  of  the  second  order,  which  are  able  to  unite  with  the       .  > 
agglutinogenic  receptors  of  the  microorganism.     It  has  been  well  es-  I/ 
tablished  that  the  number  of  such  suitable  receptors  vary  in  animals,  and 
that  different  animals  may  not  produce  serums  with  equal  agglutinating 
powers. 

Agglutinins  do  not  appear  in  the  serum  immediately  after  inocula-  '•" 
tion,  but  require  an  incubation  period  of  from  two  to  four  days  for  their 
production. 

Properties  and  Nature  of  Agglutinins. — (1)  Agglutinins  are  fairly 
resistant  substances  that  withstand  heating  to  60°  C.  for  thirty  minutes 
and  lose  their  power  only  when  heated  to  higher  temperatures.    It  is 
possible,  therefore,  to  make  a  serum  bacteriolytically  inactive  by  de-  K 
stroying  complement  at  55°  C.,  and  still  retain  its  agglutinating  power. 

(2)  They  resist  drying,  and  their  activity  is  best  preserved  in  this   I/ 
state.    They  do  not  dialyze  through  animal  membranes. 

(3)  They  are  precipitated  from  a  serum  by  magnesium  or  ammonium    «/ 
sulphates,  when  these  salts  are  used  in  proper  concentration,  and  are 
thus  closely  associated  with  the  globulin  fraction  of  serum. 

(4)  They  are  separate  and  distinct  antibodies,  and  are  not  associated 
with  bacteriolysins.     Thus  the  agglutinins  of  an  immune  serum  may  be 
lost,  destroyed,  or  absorbed  and  the  bacteriolysins  retained.     As  pre- 
viously mentioned,  the  bacteriolytic  power  of  a  serum  may  be  inhibited 
by  heating  it  to  55°  C.  for  one-half  hour  without  influencing  the  agglu- 
tinin  content,  and  during  disease  processes  the  formation  of  agglutinins 
and  that  of  bacteriolysins  are  apparently  not  parallel  processes. 

Acid  Agglutination. — Bacteria  may  be  agglutinated  by  acids,  and 
the  method  of  acid  agglutination  was  introduced  by  Michaelis1  for  the 
differentiation  of  bacterial  species  on  the  basis  that  the  hydrogen  ion 
concentration  at  which  agglutination  is  maximal  is  characteristic  for 
various  species  of  closely  allied  types.  The  results  of  considerable 
investigation  on  the  acid-agglutination  of  the  typhoid-colon  group  of 
bacilli  has  shown  that  Bacillus  typhosus  and  Bacillus  paratyphosus  are 
readily  distinguished  by  means  of  the  reaction.  Definite  differentiation 
between  the  paratyphoid  bacilli  A,  B,  and  C  have  not  been  seen  by 
Beniasch,2  Jaffe,3  Heinmann,4  and  Grote5;  Beniasch  has  also  reported 

1  Deutsch.  med.  Wochenschr.,  1911,  37,  969. 

2  Ztsch.  f.  Immunitatsf.,  brig.,  1912,  12,  268.  3  Arch.  f.  Hyg.,  1912,  76,  1. 
4  Ztsch.  f.  Immunitatsf.,  orig.,  1913,  16,  127. 

5Centralb.  f.  Bakteriol.,  etc.,  orig.,  1913,  69,  98. 


284  AGGLUTININS 

the  resistance  of  Bacillus  coli  to  acid  agglutination  at  any  hydrogen  ion 
concentration,  and,  indeed,  he  has  found  certain  strains  of  nearly  all 
species  of  bacteria  to  be  non-agglutinable  within  the  tested  reaction 
limits.  Gillespie6  found  that  pneumococci  belonging  to  the  serologic  (/ 
types  I  and  II  have,  as  a  rule,  narrow  zones  of  agglutination.  The 
optimum  hydrogen  ion  concentration  was  found  different  in  the  two 
cases,  while  other  pneumococci  had  broad  zones  or,  in  a  few  cases, 
narrow  zones  not  coincident  with  those  occupied  by  the  typical  organ- 
isms. For  a  technic  of  acid  agglutination  see  page  310. 

Mechanism  of  Agglutination. — The  true  nature  of  the  phenomenon 
of  agglutination  is  unknown,  as  is  shown  by  the  number  of  theories  ad- 
vanced. Thus — 

1.  Gruber's  idea  of  the  mechanism  of  this  phenomenon  was  that  the 
agglutinin  so  changed  the  bacterial  membrane  as  to  render  it  more 
viscous,  and  that  this  increased  viscosity  caused  the  bacteria  to  adhere 
and  form  clumps.     No  visible  changes  in  the  organisms  or  red  corpuscles 
can,  however,  be  seen. 

2.  Paltauf's  theory  is  somewhat  similar,  he  believing  that  the  agglu- 
tinogen  is  precipitated  on  the  surface  of  the  bacteria  by  union  with  the 
agglutinin,  with  the  formation  of  a  sticky  substance.     He  cites  evidence 
that  tends  to  show  that  such  substances  are  actually  thrown  out  from 
the  bacteria  during  agglutination,  as  may  be  seen  in  a  properly  stained 
preparation  in  the  form  of   a  precipitate   surrounding   the  bacterial 
cells. 

3.  The  presence  of  some  salt  is  necessary  for  the  occurrence  of  agglu- 
tination.    Bordet  found  that  if  the  salts  were  removed  from  the  serum 
and  from  the  suspension  of  bacteria  by  dialysis  and  that  the  two  were 
then  mixed,  agglutination  did  not  occur,  but  that  if  a  small  amount 
of  sodium  chlorid  was  added,  agglutination  promptly  took  place.     Ac- 
cording to  this  view,  therefore,  agglutination  is  a  phenomenon  of  molec- 
ular physics — the  agglutinin  acts  on  the  bacteria  or  other  cells  and 
prepares  them  for  agglutination  by  altering  the  relations  of  molecular 
attraction  between  them  and  the  surrounding  fluid,  the  second  phase, 
the  loss  of  motility,  clumping,  etc.,  being  brought  about  by  the  presence 
of  salt.     This  second  phase,  therefore,  would  be  a  purely  physical 
phenomenon,  the  salts  altering  the  electric  conditions  of  the  colloidal- 
like  agglutinin-bacterium  combination,  so  that  their  surface  tension  is 
increased.     To  overcome  this  the  particles  adhere  together,  presenting 
in  a  clump  less  surface  tension  than  if  they  remained  as  individual  par- 

1  Jour.  Exper.  Med.,  1914,  19,  28 


DIFFERENTIATING    BETWEEN   A    MIXED   AND    SINGLE    INFECTION    285 

tides.  Bordet  cites  the  precipitation  of  clay  as  an  analogous  case:  if 
a  little  salt  is  added  to  a  fine  emulsion  of  potters'  clay  in  distilled  water, 
the  clay  immediately  clumps  and  falls  to  the  bottom,  the  resemblance 
between  these  flakes  and  the  clump  of  agglutinated  bacteria  being  very 
striking. 

Specificity  of  Agglutinins. — For  a  time  after  their  discovery  the  ag- 
glutinins  were  regarded  as  strictly  specific,  i.  e.,  a  typhoid-immune  serum 
would  agglutinate  only  typhoid  bacilli  and  no  others.  Gruber  early 
pointed  out  that  an  immune  serum  will  frequently  agglutinate  other 
closely  related  organisms,  although  not  usually  to  so  high  a  degree. 

Group  or  partial  agglutinins,  therefore,  refer  to  the  presence  in  a  serum 
of  certain  agglutinins  that  agglutinate  certain  other  microorganisms 
that  are  morphologically,  biologically,  and  often  pathogenetically 
closely  related  to  the  homologous  microorganism  (the  bacterium  causing 
the  infection  or  used  in  artificial  immunization).  For  example,  a  ty- 
phoid-immune serum  possesses,  besides  its  greatly  increased  aggluti- 
nating power  for  Bacillus  typnosus,  some  agglutinin  for  Bacillus  para- 
typhosus,  notably  above  that  of  normal  serum.  This  is  explained  by  the 
very  close  biologic  relationship  of  these  bacteria,  together  with  the  fact 
that  the  agglutinin-producing  substance  (agglutinogen)  is  a  complex 
and  not  a  single  chemical  substance.  This  has  been  explained  by  Dur- 
ham in  the  following  example :  If  the  typhoid  agglutinogen  is  composed 
of  various  elements,  A,  B,  C,  D,  it  is  conceivable  that  the  closely  related 
paratyphoids  might  contain  one  or  more  of  these  four  agglutinogens, 
and,  therefore,  the  agglutinating  power  of  the  typhoid  serum  for  a  para- 
typhoid bacillus,  though  not  so  great  as  on  the  typhoid  bacillus,  is  still 
considerable.  Accordingly,  in  an  infection  with  one  microorganism 
a  specific  agglutinin  will  be  formed  for  that  microorganism,  and  group 
agglutinins  for  other  more  or  less  allied  microorganisms,  and  conse- 
quently the  specificity  of  the  agglutinating  reaction  depends  upon  the 
principle  of  dilution,  the  specific  agglutinin  being  present  in  largest 
amount  and  operative  in  dilutions  above  the  range  of  the  group  agglu- 
tinins. 

Absorption  Methods  for  Differentiating  Between  a  Mixed  and  a 
Single  Infection. — In  1902  Castellani  discovered  that  if  the  serum  of  an 
animal  immunized  against  a  certain  microorganism  contains  that 
microorganism  in  large  numbers,  the  serum  will  lose  its  agglutinating 
power  not  only  for  that  microorganism,  but  also  for  all  other  varieties 
on  which  it  formerly  acted.  If,  however,  the  serum  contains  the  organ- 
ism corresponding  to  the  group  agglutinins,  the  agglutinating  power  of 
the  serum  for  the  homologous  organism  is  reduced  but  little  or  not  at  all. 


286  AGGLUTININS 

In  a  mixed  infection  due  to  two  or  more  varieties  of  bacteria  there 
iwill  be  specific  agglutinins  for  each  of  the  microorganisms,  and  group 
agglutinins  for  each  of  them  as  well.  If  the  immune  serum  is  saturated 
with  one  of  these  varieties  its  chief  or  major  agglutinins  and  some  or  all 
of  the  group  agglutinins  will  be  removed,  but  the  major  agglutinin  of  the 
second  species  will  remain.  On  the  addition  of  the  second  bacterium  to 
the  immune  serum  agglutination  occurs  and  its  agglutinin  is  absorbed. 
Park,  who  has  carefully  investigated  this  subject,  finds  that  the  absorp- 
tion method  proves  that  when  one  variety  of  bacteria  removes  all  agglu- 
tinins for  a  second,  the  agglutinins  in  question  were  not  produced  by  the 
second  variety. 

Hemagglutinins. — Agglutination,  like  other  immunity  reactions,  is  a 
manifestation  of  broad  biologic  laws  and  is  not  limited  to  bacteria.  As 
hemolysins  are  produced  by  the  injection  of  an  animal  with  red  blood- 
corpuscles  from  another  species,  so  agglutinins  that  agglutinate  the  red 
blood-corpuscles  may  be  developed  at  the  same  time.  When  a  serum 
containing  hemagglutinin  is  added  to  a  suspension  of  the  corresponding 
red  blood-corpuscles  contained  in  a  test-tube,  it  causes  these  to  collect 
into  clumps  and  flakes  and  sink  to  the  bottom,  just  as  a  typhoid  immune 
serum  agglutinates  typhoid  bacilli.  These  clumps  are  broken  up  with 
some  difficulty,  and  may  interfere  with  hemolytic  reactions.  They  are 
especially  to  be  observed  in  antihuman  hemolytic  serums  when  agglu- 
tination may  be  so  marked  as  to  prevent  hemolysis  unless  the  tubes  are 
frequently  and  vigorously  shaken. 

Small  amounts  of  normal  hemagglutinins  may  be  found.  Of  par- 
ticular practical  importance  are  those  for  animals  of  the  same  species, 
the  so-called  isohemagglutinins. 

Isohemagglutinins  and  their  Relation  to  Blood  Transfusion. — Isohem- 
agglutinins were  discovered  independently  by  Landsteiner,1  and  Shattuck 
in  1900,  and  have  been  studied  quite  extensively  by  Hektoen,  Otten- 
burg,2  Moss,3  Gay,  Brem,4  and  others.  At  first  the  occurrence  of  isoag- 
glutination  was  regarded  as  of  pathologic  signifiance,  but  later  researches 
showed  that  they  may  be  found  in  a  large  percentage  of  normal  bloods. 
According  to  Landsteiner,  human  bloods  may  be  divided  into  three 
groups;  the  fourth  group  was  discovered  independently  by  several  ob- 
servers. 

Group  1:  Here  the  corpuscles  are  not  agglutinated  by  any  human 

1  Wien.  klin.  Wochenschr.,  1901,  xiv,  1132; 

2  Jour.  Exper.  Med.,  1911,  13,  425;  ibid.,  536. 

3  Bull.  Johns  Hopkins  Hosp.,  1910,  21,  63. 

4  Jour.  Amer.  Med.  Assoc.,  1916,  67,  190. 


HEMAGGLUTININS  287 

serum,  whereas  the  serums  agglutinate  the  corpuscles  of  the  other  groups. 
This  group  includes  about  50  per  cent,  of  all  persons. 

Group  2 :  In  this  group  the  corpuscles  are  agglutinated  by  the  serums 
of  groups  1  and  3  only,  whereas  the  serums  agglutinate  the  corpuscles  of 
groups  3  and  4,  but  not  of  Group  1. 

Group  3:  The  corpuscles  are  agglutinated  by  all  other  serums,  and 
the  serums  agglutinate  the  corpuscles  of  groups  2  and  4,  but  not  of 
Group  1. 

Group  4:  The  corpuscles  are  agglutinated  by  the  sera  of  all  other 
groups,  but  the  serums  are  unable  to  agglutinate  any  human  corpuscles. 
These  are  quite  rare. 

The  group  characteristics  are  hereditary,  and  permanent  throughout 
life. 

Moss  found  that  all  normal  and  pathologic  bloods  alike  could  be 
classified  into  four  groups  by  agglutination  tests  of  the  serums  against 
the  corpuscles.  These  groups  are: 

Group  1  (10  per  cent.) :  Serum  does  not  agglutinate  any  corpuscles. 
Corpuscles  agglutinated  by  the  serums  of  groups  2,  3,  and  4. 

Group  2  (40  per  cent.):  Serum  agglutinates  corpuscles  of  groups 
1  and  3,  not  2  or  4.  Corpuscles  agglutinated  by  serums  of  groups  3  and  4, 
not  1  or  2. 

Group  3  (7  per  cent.):    Serum  agglutinates  corpuscles  of  groups 

1  and  2,  not  3  or  4.     Corpuscles  agglutinated  by  serums  of  groups 

2  and  4,  not  1  or  3. 

Group  4  (43  per  cent.):  Serum  agglutinates  corpuscles  of  groups 
1,  2,  and  3,  not  4.  Corpuscles  not  agglutinated  by  any  serums. 

It  may  be  seen  that  no  serum  agglutinates  corpuscles  belonging 
to  its  own  group,  and  furthermore,  that  if  one  has  a  known  Group  2  or 
Group  3  blood,  he  can  determine  the  group  of  any  other  blood  by  testing 
the  known  serum  against  the  unknown  corpuscles,  and  the  unknown 
serum  against  the  known  corpuscles. 

Because  reactions  may  follow  transfusion  if  the  bloods  of  donor  and 
recipient  are  incompatible  it  is  advisable  to  apply  these  preliminary 
tests;  the  technic  is  described  on  page  311. 

With  the  increasing  number  of  blood  transfusions,  the  phenomena  of 
isoagglutination  and  isohemolysis — the  two  being  closely  related — are 
of  considerable  practical  importance,  especially  if  the  patient  is  suffering 
with  cancer,  when  the  serum  is  likely  to  be  actively  hemolytic  for  the 
donor's  corpuscles. 

In  selecting  the  donor  for  a  transfusion,  agglutination  and  hemolysis 


288  AGGLUTININS 

tests  should  always  be  made  before  operation  if  time  permits.  The 
tests  made  in  vitro  are  usually  safe  guides  as  to  conditions  existing  in 
vivo,  and  such  preliminary  tests  may  prevent  the  occurrence  of  untoward 
symptoms  associated  with  intravascular  hemolysis  or  agglutination, 
such  as  fever,  dyspnea,  edema,  and  hemoglobinuria.  As  a  rule,  the 
donor  selected  should  be  a  near  relative,  and  whenever  time  permits,  a 
Wassermann  reaction  and  the  isoagglutination  and  isohemolysin  tests 
should  be  made.  That  donor  should  be  chosen  whose  blood  shows  no 
inter-agglutination  or  hemolysis  with  the  patient's  serum  and  corpuscles. 
If  such  a  donor  cannot  be  obtained,  it  is  safer  to  use  a  person  whose  serum 
is  agglutinative  toward  the  patient's  cells  than  one  whose  cells  are 
agglutinated  by  the  patient's  serum. 

The  technic  of  these  transfusion  tests  is  given  at  the  end  of  this 
chapter. 

Non-agglutinable  Species  of  Bacteria. — Certain  species  of  bacteria, 
especially  when  freshly  isolated  from  the  animal  body,  may  prove  them- 
selves immune  to  the  action  of  agglutinins;  this  is  true  especially  of 
the  bacillus  of  Friedlander.  As  a  rule,  this  resistance  is  lost  when  the 
microorganism  is  grown  for  some  time  in  artificial  media.  In  some  in- 
stances the  typhoid  bacillus,  when  freshly  isolated  from  a  patient,  may 
resist  agglutination  until  after  it  has  passed  a  period  of  existence  on 
artificial  media.  This  variability  is  probably  due  to  some  change 
taking  place  in  the  agglutinable  substance  of  the  agglutinins  during  the 
sojourn  of  the  bacilli  in  the  animal  body,  and  possess  such  an  excess  of 
agglutinogenic  receptors  as  to  require  a  much  larger  amount  of  aggluti- 
nin  to  cause  agglutination. 

It  should  be  remembered  that  agglutinins  act  on  dead  as  well  as  on 
living  bacteria,  those  killed  by  heat,  formalin,  phenol,  etc.,  being  simi- 
larly agglutinable.  In  making  the  microscopic  test  the  use  of  dead 
bacteria  is  not  so  satisfactory  as  when  the  test  is  made  with  living  motile 
bacteria,  for  the  influence  of  the  serum  on  motility  alone  is  of  value  in 
interpreting  a  reaction. 

Variation  in  Agglutinating  Strength  of  a  Serum. — In  a  given  infection, 
such  as  typhoid  fever,  there  is  usually  a  continued  increase  in  the  amount 
of  agglutinin  in  the  blood  from  the  fourth  day  until  convalescence  is 
established,  and  then  a  decrease  occurs.  It  is  a  fact  of  practical  impor- 
tance that  the  agglutinating  power  of  a  serum  may  vary  from  day  to 
day,  so  that  it  is  very  strong  one  day  and  may  become  weak  or  disap- 
pear entirely  on  the  next  day  or  two.  Hence  the  importance  of  making 
more  than  one  test  in  a  suspicious  case  when  the  first  trial  has  been 


ROLE   OF   AGGLUTININS   IN   IMMUNITY  289 

doubtful  or  negative.  There  is  no  satisfactory  explanation  for  this 
variation,  mixed  infection,  intestinal  hemorrhage,  etc.,  being  regarded 
by  some  as  responsible  for  it. 

Conglutination. — In  1906  Bordet  and  Gay1  described  a  colloidal 
substance  in  beef  serum  heated  to  56°  C.  which  has  the  property  of  caus- 
ing a  characteristic  clumping  and  increased  lysis  of  red  blood-cells  when 
treated  with  a  heated  specific  hemolytic  serum  and  fresh  alexin  (com- 
plement). Bordet  and  Streng,2  in  later  studies  on  this  substance,  gave 
to  it  the  name  "conglutinin."  Streng3  continued  these  studies  with 
bacteria  and  found  that  a  typical  clumping  was  produced  by  the  mixture 
of  bacteria,  fresh  complement,  conglutinin,  and  a  specific  immune  serum 
from  which  the  agglutinins  had  been  removed  by  absorption.  By  dia- 
lyzing  the  beef  serum  the  conglutinin  was  shown  to  be  present  in  the 
globulin  fraction,  and  the  reaction  took  place  as  well  with  bacteria 
killed  by  heat  or  0.1  per  cent,  liquor  formaldehydi  as  with  live  organisms. 

In  a  study  of  dysentery  in  infants,  Lucas,  Fitzgerald  and  Schorer4  first 
applied  this  reaction  to  clinical  diagnosis.  They  found  it  more  sensitive 
and  specific  than  either  the  agglutination  or  fixation  test. 

In  their  work  cultures  of  the  Flexner  and  Shiga  dysentery  bacilli 
treated  with  0.1  per  cent,  liquor  formaldehydi  were  used.  They  con- 
clude that  in  the  conglutination  test  we  have  a  means  of  diagnosis  far 
superior  to  any  other  form. 

Swift  and  Thro5  did  not  find  the  conglutination  reaction  of  much 
greater  value  in  the  differentiation  of  various  strains  of  streptococci  than 
the  agglutination  reaction. 

The  technic  of  this  reaction  is  given  on  page  307. 

R8le  of  Agglutinins  in  Immunity. — The  agglutinins  were  formerly 
regarded  as  possessing  a  true  protective  and  curative  power.  It  has 
previously  been  mentioned  that  bacteria  may  be  grown  in  a  specific 
agglutinating  serum,  and  cultures  made  of  agglutinated  bacteria  show 
them  to  be  fully  alive  and  as  virulent  as  before  agglutination  took  place. 
In  certain  cases  agglutinins  for  a  microorganism  may  be  entirely  absent, 
and  yet  the  animal  enjoy  an  immunity.  Bacteria  that  have  been  acted 
upon  by  an  agglutinin  are  apparently  not  altered  in  appearance,  viabil- 
ity, or  virulence. 

Many  observations  tend  to  show  that  the  agglutinating  power  of  a 

1  Am.  de  PInst.  Pasteur,  1906,  xx,  467. 

2  Centralb.  f.  Bakteriol.,  1909,  xlix,  260. 

3  Centralb.  f.  Bakteriol.,  1909,  1,  47. 

4  Jour.  Amer.  Med.  Assoc.,  1910,  Ivi,  441. 

5  Archiv.  Int.  Med.,  1911,  7,  24. 
19 


AGGLUTININS 

serum  gives  no  indication  of  the  degree  of  immunity  that  exists.  For 
instance,  relapses  may  occur  in  typhoid  fever  at  a  time  when  the  agglu- 
tinating power  of  the  patient's  blood  is  at  its  highest. 

At  present  agglutinins  are  regarded  as  playing  a  subsidiary  role  in 
immunity,  their  presence  being  of  diagnostic  value,  and  an  indication  of 
the  presence  of  more  important  factors,  and  as  an  aid  to  bacteriolysis, 
and  phagocytosis.. 

As  shown  by  Bull1  experimentally,  agglutination  may  occur  in  vivo, 
and  the  power  of  the  blood  to  cause  agglutination  determines,  in  large 
measure,  whether  after  their  direct  introduction  in  an  experimental  way 
the  bacteria  are  to  be  removed  promptly  from  the  circulation  and  bac- 
teremia  avoided,  or  whether  they  are  to  remain  and  produce  bacteremia. 
Microorganisms  which  are  not  agglutinated  in  the  normal  rabbit  may 
be  made  to  do  so  by  the  intravenous  injection  of  homologous  immune 
serum,  and  this  probably  explains  the  rapid  disappearance  of  pneumo- 
cocci  from  the  blood  following  the  intravenous  injections  of  homologous 
antipneumococcus  serum.  The  bacterial  clumps  accumulate  in  the 
organs  in  which  they  are  phagocyted.  In  this  wray  it  appears  that  ag- 
glutinins, opsonins,  and  phagocytosis  are  closely  related  and  probably 
exert  an  important  role  in  resistance  to.  and  recovery  from,  infection. 

PRACTICAL  APPLICATIONS 

The  agglutination  reaction  is  used  for  the  following  purposes: 
1.  For  the  diagnosis  of  disease,  by  identifying  the  bacterial  infection 
from  which  the  patient  is  suffering.  To  do  this  satisfactorily  we  must 
have  on  hand  stock  cultures  of  bacteria,  and  test  the  patient's  serum  for 
agglutinins  for  these  bacteria.  For  instance,  if  a  patient  presents  symp- 
toms of  typhoid  fever,  the  serum  is  tested  for  typhoid  agglutinins;  if 
the  reaction  is  very  weak  or  negative  and  continues  so,  the  serum  is 
further  tested  for  agglutinins  for  Bacillus  paratyphosus  A  and  B. 

In  typhoid  fever  the  Gruber-Widal  reaction  may  be  positive  as  early 
as  the  third  day;  usually,  however,  the  positive  reaction  is  obtained 
somewhat  later — about  the  seventh  or  the  eighth  day.  A  day  or  so 
earlier  the  bacilli  used  in  making  the  test  may  be  seen  to  lose  their  mo- 
tility,  and  two  or  three  may  form  a  loose  clump.  This  is  the  doubtful 
reaction,  and  it  is  well  to  test  every  day  or  every  other  day  until  a  de- 
cisive reaction  is  obtained. 

According  to  Park,  "  about  20  per  cent,  of  typhoid  infections  give 

1  Jour.  Exper.  Med.,  1915,  22,  484;  ibid.,  1916,  24,  25. 


PRACTICAL    APPLICATIONS  291 

positive  reactions  in  the  first  week;  about  60  per  cent,  in  the  second 
week;  about  80  per  cent,  in  the  third  week;  about  90  per  cent,  in  the 
fourth  week,  and  about  75  per  cent,  in  the  second  month  of  the  disease." 
In  about  90  to  95  per  cent,  of  cases  in  which  repeated  examinations  are 
made  a  positive  reaction  is  to  be  found  at  some  time  during  the  patient's 
illness. 

Occasionally  the  reaction  appears  first  during  the  stage  of  convales- 
cence, and  at  times  it  may  even  be  absent,  the  diagnosis  being  confirmed 
by  cultivating  typhoid  bacilli  from  the  blood.  The  possibility  of  a  given 
case  reacting  strongly  one  day  and  weakly  or  entirely  negative  a  day  or 
so  later  has  been  emphasized  elsewhere. 

Usually  the  reaction  is  strongest  during  convalescence,  remains  posi- 
tive for  several  weeks,  and  then  gradually  returns  to  the  normal.  Occa- 
sionally the  reaction  remains  positive  for  months  or  even  years  after  the 
attack  of  typhoid  fever — many  such  cases  are  " carriers"  and  harbor  the 
bacilli  in  the  gall-bladder,  although  the  person  appears  to  be  quite  well. 

Only  very  rarely  does  normal  serum  immediately  agglutinate  typhoid 
bacilli  in  a  dilution  higher  than  1:10;  where  a  time  limit  of  one  to 
two  hours  is  given,  a  few  may  show  some  agglutination  in  dilutions  up 
to  1 :  30. 

If  the  typhoid  bacillus  is  agglutinated  by  the  patient's  serum  in  a 
dilution  of  1:100,  or  at  least  1:40,  the  Widal  reaction  may  be  regarded 
as  positive.  It  is  not  safe  to  use  lower  dilutions,  as  occasionally  the 
serum  of  healthy  persons  may  agglutinate  Bacillus  typhosus  in  dilutions 
up  to  1:30.  Due  care  must  be  exercised  not  to  mistake  a  pseudo- 
reaction  about  detritus  for  true  agglutination. 

Positive  reactions  are  occasionally  obtained  in  other  diseases — acute 
miliary  tuberculosis,  malaria,  malignant  endocarditis,  and  pneumonia. 
It  is  also  well  to  bear  in  mind  the  possibility  of  a  patient  having  been 
vaccinated  against  typhoid  at  some  early  date,  with  resulting  agglutinin 
formation. 

Owing  to  the  similarity  of  symptoms  an  infection  with  Bacillus  para- 
typhosus  A  and  B  may  be  difficult  to  distinguish  from  typhoid  fever. 
This  difficulty  is  increased  by  the  confusion  of  the  Widal  reaction 
owing  to  the  presence  of  group  agglutinins  in  the  serum  if  proper  dilu- 
tion is  not  practised.  Bacillus  A  and  Bacillus  B  are  not  identical  in  their 
agglutinable  properties,  the  latter  being  more  closely  related  to  the 
typhoid  bacillus  than  the  former.  In  this  country  Bacillus  paratyphosus 
is  usually  held  responsible  for  paratyphoid  fever.  Conclusions  should 
not  be  drawn  until  tests  have  been  made  with  both  strains  of  the  para- 


292  AGGLUTININS 

typhoid  bacillus  and  with  the  typhoid  bacillus.  In  case  of  mixed  in- 
fection the  absorption  method  of  Castellani  will  serve  to  clear  up  the 
diagnosis. 

In  dysentery  the  agglutination  reaction  with  the  serum  of  patients 
shows  great  variability.  In  spite  of  the  presence  of  bacilli  in  the  feces 
ithe  reaction  is  sometimes  absent,  often  disappears  rapidly  during  con- 
valescence, and  rarely  is  as  high  as  in  typhoid  fever.  The  tests  should 
always  be  performed  with  both  the  "Flexner"  and  "Shiga"  types  of 
bacilli,  as  the  two  do  not  possess  identical  agglutinable  properties,  and 
either  may  be  the  cause  of  infection  in  a  given  case.  The  absence  of  the 
reaction  does  not  exclude  a  dysenteric  infection. 

In  cholera  the  agglutination  test  has  so  far  proved  of  doubtful  aid  in 
establishing  a  diagnosis  of  the  disease.  However,  for  the  purpose  of 
recognizing  bacilli  isolated  from  the  feces  of  suspicious  cases  the  reaction 
with  known  immune  serum  is  of  great  value. 

In  cerebrospinal  meningitis  the  agglutination  occurs  within  an  hour 
in  dilutions  of  1 : 10.  It  is  seldom  that  the  patient's  serum  agglutinates 
in  a  dilution  higher  than  1 : 50. 

In  tuberculosis  the  agglutination  reaction  has  no  value  as  a  diagnostic 
procedure.  Koch  recommended  the  agglutination  test  for  the  estima- 
tion of  the  degree  of  immunity  conferred  by  tuberculin  treatment.  As 
pointed  out  elsewhere,  agglutinins  have  apparently  no  antimicrobic 
influence,  but,  as  with  typhoid  vaccination,  may  indicate  the  degree  of 
reaction  and  the  presence  of  other  antibodies. 

Many  strains  of  tubercle  bacilli  are  almost  non-agglutinable.  The 
preparation  of  a  homogeneous  emulsion  is  not  easily  made,  and  the  results 
are  likely  to  be  confusing  and  contradictory. 

In  plague  the  agglutination  reaction  becomes  quite  marked  about 
the  ninth  day  of  the  disease — too  late,  however,  to  be  of  much  practical 
value  in  diagnosis.  It  is  occasionally  useful,  however,  for  deciding 
whether  a  patient  in  the  convalescent  stage  has  really  suffered  from  the 
disease. 

In  Malta  fever  the  agglutination  reaction  is  of  considerable  value  in 
making  the  diagnosis. 

In  pneumonia  the  reaction  is  of  value  in  rapidly  differentiating  pneu- 
mococci  and  as  an  aid  in  specific  serum  therapy  (see  page  308),  and  it 
may  also  be  of  aid  in  making  the  diagnosis  of  infection  with  Bacillus 
enteritidis  and  Bacillus  botulinus. 

Agglutinins  for  Treponema  pallidum  by  the  sera  of  rabbits  injected 
with  a  living  and  heat-killed  culture  furnished  by  Noguchi,  were  first 


PRACTICAL   APPLICATIONS  293 

described  by  Kolmer1;  Nakano,2  Kissmeyer,3  and  Zinsser  and  Hopkins4 
have  also  described  the  agglutination  of  culture  pallidum  by  immune 
serum.  Kolmer,  Broad  well  and  Matsunami5  found  that  equal  parts  of 
normal  human  serum  and  pallidum  culture  furnished  by  Zinsser,  may 
result  in  partial  agglutination,  whereas  with  sera  of  syphilitics  in  the 
later  stages,  agglutination  in  dilutions  of  1  : 5  and  higher  occurred 
with  about  84  per  cent,  of  sera,  but  that  further  studies  are  neces- 
sary to  establish  the  practical  value  of  agglutination  in  the  diagnosis 
of  syphilis. 

In  pertussis  agglutination  tests  have  been  found  of  some  value  in 
clinical  diagnosis  by  Wollstein,6  Frankel,7  Seiffert,8  and  Arnheim;  Bordet9 
finds  it  of  value  only  when  the  microorganism  is  freshly  isolated  from 
human  sputum  and  grown  on  rich  blood  medium .  According  to  Povitsky 10 
and  Worth11  a  dilution  of  serum  not  less  than  1  : 200  is  necessary  for 
a  practical  positive  diagnosis  of  pertussis,  as  normal  human  serum  may 
agglutinate  in  dilutions  up  to  1  : 100. 

In  veterinary  practice  agglutination  reactions  are  of  value  in  the 
diagnosis  of  glanders,  infected  horses  reacting  in  some  instances  to  dilu- 
tions as  high  as  1:2000.  For  diagnostic  purposes  the  agglutination 
test  in  glanders  must  be  in  dilutions  higher  than  1:800.  A  positive 
reaction  in  dilutions  of  1:1000  is  regarded  as  suggestive,  and  is  con- 
trolled by  a  complement-fixation  test;  agglutination  in  dilutions  of 
1 : 1500  practically  always  indicates  an  infection.  The  complement-fixa- 
tion test,  however,  is  a  better  diagnostic  reaction. 

2.  Agglutination  reactions  are  also  of  value  as  an  aid  to  the  identi- 
fication of  a  microorganism  that  has  been  cultivated  from  a  patient. 
For  this  purpose  we  must  have  on  hand  various  standard  immune 
serums.  For  example,  if  a  bacillus  resembling  the  typhoid  bacillus  is 
isolated  from  the  feces  of  a  patient,  the  diagnosis  may  be  aided  by  a 
positive  agglutination  reaction  with  typhoid  immune  serum. 

1  Jour.  Exper.  Med.,  1913,  xviii,  18. 

2  Archiv.  Dermat.  u.  Syph.,  1913,  cxvi,  265. 

3  Deutsch.  Med.  Wchnschr.,  1915,  xli,  306. 

4  Jour.  Exper.  Med.,  1915,  xxi,  576. 

5  Jour.  Exper.  Med.,  1916,  xxiv,  333. 

6  Jour.  Exper.  Med.,  1909,  xi,  41. 

7  Munch.  Med.  Wchnschr.,  1908,  Iv,  1683. 
*  Munch.  Med.  Wchnschr.,  1909,  1561. 

9  Berl.  klin.  Wchnschr.,  1908,  xlv,  1453. 

10  Centralb.  f .  Bakteriol.,  orig.,  1912,  Ixvi,  276. 

11  Archiv.  Int.  Med.,  1916,  xvii,  279. 


294  AGGLUTININS 

The  "  group  agglutinins"  constitute  a  source  of  difficulty  in  making 
the  differentiation  among  the  numerous  members  of  a  group  of  micro- 
organisms, but  if  a  highly  potent  agglutinating  serum  is  used  and  the 
test  is  carried  to  the  point  of  determining  the  highest  dilution  that  will 
agglutinate  the  bacteria,  it  will  in  most  cases  be  possible  to  differentiate 
the  variously  allied  microorganisms  by  this  test. 

In  conducting  these  reactions  it  is  best  to  use  a  macroscopic  method, 
and  the  agglutinating  serum  used  must  have  been  previously  titrated 
against  an  easily  agglutinable  and  known  strain  of  the  microorganism 
in  question 

In  this  connection  it  is  well  to  remember  that  freshly  isolated  cultures 
of  a  microorganism  may  be  not  at  all  or  but  very  slightly  agglutinable. 
Thus  colonies  of  typhoid  bacilli  found  in  feces  or  in  an  abscess  may,  if 
picked  from  a  plate,  resist  agglutination  until  subcultured  several  times 
in  artificial  media. 

The  agglutination  test  has  great  value  as  a  mode  of  differentiating 
among  the  members  of  the  typhoid-colon  group  of  bacilli.  In  the  diag- 
nosis of  cholera,  suspicious  bacilli  isolated  from  the  feces  may  be  tested 
with  a  known  cholera  immune  serum,  and  the  bacteriologic  diagnosis 
thus  be  greatly  facilitated.  The  test  also  has  some  value  in  making  a 
biologic  differentiation  between  meningococci  and  gonococci,  and  also 
between  other  groups  of  bacteria. 

3.  Agglutination  tests  are  of  value  in  determining  whether,  in  a  case 
in  which  more  than  one  microorganism  has  been  cultivated,  the  condi- 
tion at  hand  is  a  single  or  a  mixed  infection.      The  absorption  agglu- 
tinin  test  is  made  with  the  patient's  serum,  and  the  cultures  are  isolated 
from  the  patient. 

4.  'Agglutination  tests  are  also  of  value  for  measuring  the  immuniz- 
ing response  that  a  patient  is  making  to  his  infection  or  to  artificial 
immunization.     Thus  the  test  is  of  some  value  in  determining  the  re- 
sponse to  inoculation  with  typhoid  vaccine,  although  it  is  probable 
that  the  agglutinin  itself  does  not  possess  true  antimicrobic  properties. 


THE  AGGLUTINATION  REACTION 

The  value  of  the  agglutination  test  in  the  diagnosis  of  disease  is 
limited  chiefly  to  typhoid  and  paratyphoid  fevers;  and,  in  a  less  degree, 
to  cerebrospinal  meningitis  and  bacillary  dysentery.  It  is  especially 
useful  as  a  practical  test  in  the  diagnosis  of  obscure  and  atypical  cases  of 
typhoid  fever  and  also  in  the  diagnosis  of  " typhoid  carriers." 


THE    AGGLUTINATION   REACTION  295 

Two  methods  may  be  employed: 

1.  The  microscopic  method  which  is  generally  employed  where  the 
Gruber-Widal  reaction  for  typhoid  fever  has  been  employed,  as  the  re- 
action is  quickly  done  and  requires  but  a  small  amount  of  blood. 

This  test  is  usually  performed  with  serum  separated  from  the  clot 
and  in  various  dilutions  (wet  method) .  The  test  may  also  be  performed 
with  dried  blood  (dry  method),  the  agglutinins  being  preserved  and  re- 
dissolved  with  a  diluent.  The  technic  of  the  latter  method  is  very 
simple.  The  blood  is  easily  collected  and  may  be  sent  for  long  dis- 
tances, and  for  these  reasons  the  method  has  been  adopted  by  many 
boards  of  health. 

2.  The  macroscopic  method  is  that  generally  preferred  if  sufficient 
blood  is  on  hand,  and  is  the  method  of  choice  in  scientific  research.     Ab- 
sorption tests  must  be  performed  with  the  macroscopic  technic. 

Requisites  for  Conducting  Agglutination  Reactions. — (a)  Bacterial 
Emulsion. — This  may  be  prepared  by  growing  the  microorganism  in 
broth.  Solid  media  may  be  used,  and  the  culture  washed  off  with  sterile 
salt  solution  and  emulsified. 

(1)  The  bacterial  emulsion  should  be  prepared  of  young  cultures, 
should  be  homogeneous  and  free  from  clumps,  and  of  such  density  as  to 
furnish  a  sufficient  number  of  microorganisms  to  give  the  reaction  (Fig. 
81). 

(2)  For  the  ordinary  microscopic  Widal  test,  eighteen  to  twenty- 
four  hour  bouillon  cultures  of  Bacillus  typhosus,  Bacillus  coli,  and  Bacil- 
lus paratyphosus  yield  uniform  and  satisfactory  results.     An  old  lab- 
oratory culture — one  that  is  known  to  be  agglutinable — should  be  used. 
Broth  cultures  should  be  cultivated  at  a  temperature  lower  than  body 
heat,  in  order  that  long  motile  forms  may  be  secured.     During  the  sum- 
mer and  early  autumn  months  the  culture  can  be  grown  at  room  tem- 
perature ;  during  the  winter,  on  top  of  the  incubator. 

Thick  cultures  are  unsatisfactory  for  making  the  microscopic  test, 
as  there  is  always  some  false  clumping  and  motility  is  not  well  marked 
(Fig.  82). 

When  these  tests  are  done  routinely,  it  is  good  practice  to  subculture 
in  broth  every  day  in  order  that  a  satisfactory  culture  may  always  be 
on  hand.  When  performed  at  irregular  intervals,  a  broth  culture  can 
be  prepared  from  a  stock  agar  culture  and  the  test  performed  twenty- 
four  hours  later. 

(3)  Emulsions  may  be  prepared  of  young  cultures,  on  solid  media  by 
removing  portions  of  the  growth  with  a  platinum  loop  and  emulsifying 


296 


AGGLUTININS 


in  a  diluent,  such  as  normal  salt  solution  or  broth.  This  may  be  per- 
formed by  placing  the  diluent  in  a  test-tube  and  rubbing  the  loop  over 
the  glass  just  at  the  margin  of  the  fluid,  the  bacteria  being  gradually 
emulsified  and  floated  into  the  diluent.  When  larger  quantities  of  emul- 
sion are  desired,  as  for  making  the  macroscopic  test,  5  c.c.  of  diluent  may 
be  poured  upon  the  culture  and  the  growth  washed  off  with  the  aid  of 
the  platinum  loop.  The  emulsion  is  gently  shaken  and  removed  to  a 
second  tube,  when  unresolved  bacterial  clumps  will  sink  to  the  bottom. 
In  other  cases  the  emulsion  may  be  centrifuged  for  a  short  time  or  fil- 
tered through  sterile  filter-paper.  Sufficient  salt  solution  is  added  to 


FIG.  81. — A  SATISFACTORY  CULTURE  FOR 
THE  MICROSCOPIC  AGGLUTINATION 
REACTION.  X  430. 

This  shows  a  satisfactory  culture  of 
the  proper  density  and  free  of  clumps  of 
bacilli.  (Twenty-four  hour  culture  of 
Bacillus  typhosus  grown  at  room  tem- 
perature.) 


FIG.  82. — AN  UNSATISFACTORY  CULTURE 
FOR  THE  MICROSCOPIC  AGGLUTINA- 
TION REACTION.  X  430. 

The  culture  is  rather  too  dense  and 
shows  considerable  spontaneous  or  false 
agglutination  of  the  bacilli.  (Twenty- 
four  hour  culture  of  Bacillus  typhosus 
grown  at  37°  C.) 


give  the  emulsion  a  density  equal  to  that  of  a  rich  twenty-four-hour 
bouillon  culture. 

A  permanent  suspension  of  microorganisms  for  the  macroscopic 
test  may  be  prepared  in  the  following  manner  (Oxford  University) : 

The  bacillus  (B.  typhosus,  B.  paratyphosus,  etc.)  is  grown  for  twenty- 
four  hours  at  37°  C.  iri  ordinary  veal  peptone  bouillon  in  large  Erlen- 
meyer  flasks  partly  filled  (1  liter  of  bouillon  in  a  one  and  a  half  liter 
flask). 

Before  use  the  flasks  of  bouillon  are  sterilized  in  the  autoclave  at 


THE    AGGLUTINATION    REACTION  297 

115°C.  for  not  more  than  fifteen  minutes,  and  are  then  tested  for  sterility 
by  incubation  at  37°  C.  for  forty-eight  hours. 

They  are  inoculated  with  a  few  drops  each  from  a  twenty-  to  twenty- 
four-hour  old  bouillon  culture  of  the  bacillus  (B.  typhosus  or  B.  para- 
typhosus,  etc.). 

The  culture  used  should  be  one  which  has  been  subcultivated  daily 
in  bouillon  for  one  or  two  weeks  (or  longer).  This  continued  subculti- 
vation  has  the  effect  of  increasing  its  agglutinability  and  diminishing 
any  tendency  to  spontaneous  agglutination. 

At  the  end  of  twenty  to  twenty-four  hours'  growth  at  37°  C.  the 
flasks  are  well  shaken,  arid  to  each  is  added  0.1  per  cent.  (1  c.c.  per  liter) 
of  commercial  (40  per  cent.)  formalin.  They  are  again  shaken  and 
placed  in  a  cold  chamber  in  the  dark  at  about  2°  C. 

At  intervals  on  the  same  day  and  on  subsequent  days  for  four  or 
five  days  the  flasks  are  again  thoroughly  shaken  and  replaced  at  once 
in  the  cold  chamber. 

After  three  or  four  days  they  will  be  found  to  be  absolutely  sterilized. 
Should  it  happen  that  the  bacterial  suspension  is  not  entirely  homo- 
geneous it  may  be  shaken  for  some  hours  in  a  mechanical  shaker,  or 
may  finally  be  filtered  through  sterile  cotton-wool. 

(4)  To  emulsify  a  culture  of  the  plague  bacillus  or  any  other  micro- 
organism that  displays  a  strong  tendency  to  undergo  "spontaneous" 
agglutination,  distilled  water  or  1  : 1000  salt  solution  should  be  used. 

(5)  In  the  case  of  a  culture  of  tubercle  bacillus,  the  growth  can  be 
resolved  into  its  elements  by  prolonged  trituration  in  normal  salt  solution, 
and  any  residue  or  unresolved  clumps  removed  by  centrifugalization. 
A  less  laborious  and  dangerous  method  is  to  use  the  tubercle  powder  of 
Koch,  which  is  obtained  by  reducing  dried  tubercle  cultures  to  a  fine 
powder  by  machinery.     The  powder  may  be  made  up  into  a  suitable 
suspension  by  rubbing  it  in  a  mortar  with  normal  salt  solution. 

(6)  When  it  is  necessary  to  work  with  highly  dangerous  microorgan- 
isms, or  to  operate  from  day  to  day  with  the  same  bacterial  suspension, 
one  may  employ  suspensions  that  have  been  heated  for  one  hour  to  60° 
C.,  or  suspensions  in  salt  solution  to  which  1  per  cent,  formalin  has  been 
added.     These  will  keep  well  in  the  refrigerator,  but  the  sediment  of 
dead  bacteria  must  be  well  shaken  before  it  is  used. 

(6)  Serum. — Serum  should  preferably  be  fresh,  clear,  and  free  from 
corpuscles. 


298  AGGLUTININS 

(1)  For  the  microscopic  "wet"  method,  ample  serum  is  furnished  from 
the  blood  contained  in  the  ordinary  Wright  capsule  (see  p.  33). 

(2)  For  the  microscopic  "dry  method/'  blood  is  secured  by  pricking 
the  finger  or  lobe  of  the  ear  and  collecting  a  few  drops  of  blood  upon 
aluminum  foil,  on  a  clean  glass  slide,  or  on  partially  glazed  paper.     The 
blood  must  not  be  heated  to  hasten  drying,  or  agglutinins  may  be  destroyed. 
Smears  on  aluminum  foil  and  on  glass  slides  are  to  be  preferred  to  those 
on  paper,  as  the  blood  can  be  moistened  and  portions  removed  without 
the  likelihood  of  transferring  extraneous  material,  such  as  paper  fiber. 
While  there  are  certain  objections  to  this  method  to  be  pointed  out  later, 
yet  practical  experience  has  demonstrated  its  value,  as  the  serum  does 
not  readily  deteriorate  or  become  contaminated  with  bacteria,  and  the 
ease  with  which  blood  may  be  collected  and  mailed  recommends  the 
process  for  board  of  health  laboratories. 

(3)  For  the  macroscopic  test  larger  amounts  of  serum  are  needed. 
Sufficient  blood  is  easily  obtained  by  pricking  the  finger  deeply  and 
collecting  several  cubic  centimeters  in  a  small  test-tube. 

(4)  Because  normal  agglutinins  may  be  active  in  dilutions  as  high  as 
1 : 30,  for  diagnostic  tests  in  typhoid  fever  the  serum  should  not  be  di- 
luted lower  than  1 : 40.     For  routine  work  dilutions  of  1 : 50  and  1 : 100 
are  well  adapted  for  the  microscopic  test.     Dilutions  of  1:40  and  1:80 
are   readily   made  with  the  white  corpuscle  pipet  and   are   equally 
useful. 

With  the  macroscopic  technic,  dilutions  of  from  1:20  up  to  any 
dilution  are  readily  made  in  appropriate  test-tubes. 

Precautions. — In  bacteriologic  technic  due  care  should  be  observed 
to  avoid  contamination  and  possible  infection  when  working  with  living 
cultures. 

(a)  Agglutinated  bacteria  are  not  necessarily  dead,  and  hanging-drop 
preparations,  test-tubes,  etc.,  should  be  immersed  in  1  per  cent,  formalin 
before  cleansing. 

(6)  The  working  table  or  desk  and  the  hands  should  be  washed  with 
a  solution  of  lysol  or  1  per  cent,  formalin  after  the  reactions  have  been 
made  and  the  work  completed. 

(c)  Early  in  typhoid  fever  the  bacillus  may  be  present  in  the  blood, 
and  consequently  due  care  should  be  exercised  in  handling  it,  in  diluting 
the  serum,  and  in  the  disposal  of  the  clot. 


THE   AGGLUTINATION   REACTION  299 

TECHNIC  OF  THE  MICROSCOPIC  AGGLUTINATION  TEST  (WET  METHOD).— THE 
WIDAL  REACTION  IN  TYPHOID  FEVER 

(1)  Dilute  the  patient's  serum  by  placing  one  drop  from  a  capillary 
pipet  in  a  small  watch-glass  and  adding  19  drops  of  normal  salt  solution. 
This  gives  a  dilution  of  1  :  20.     Mix  thoroughly. 

(2)  With  a  3  or  4  mm.  platinum  loop  place  a  drop  on  a  clean  cover- 
glass  that  is  sufficiently  thin  to  permit  the  use  of  an  oil-immersion  lens. 
The  loop  is  better  than  a  capillary  pipet  because  the  drop  it  gives  is 
smaller,  and  when  it  is  later  diluted  with  an  equal  quantity  of  bacterial 
emulsion,  it  is  not  too  large  and  is  easily  manipulated. 

(3)  With  the  same  sterilized  platinum  loop  add  one  loopful  of  a 
twenty-four-hour  broth  culture  of  Bacillus  typhosus  to  the  drop  of 
diluted  serum  on  the  cover-glass.     Mix  gently  and  without  spreading  the 
drop.     This  gives  a  final  dilution  of  1 :  40. 

(4)  Edge  a  hanging-drop  slide  with  vaselin,  and  invert  the  cover- 
glass  slide  over  the  hollow  portion  in  such  a  manner  that  the  drop  will 
be  suspended  in  its  center.     Care  must  be  exercised  not  to  spread  the 
drop,  for  if  this  occurs  and  the  fluid  flows  around  the  margins  of  the 
chamber  a  new  preparation  must  be  made.     Inspect  the  slide,  and  add 
vaselin,  if  necessary,  until  it  is  sealed  tightly.     By  means  of  a  grease 
pencil  label  the  slide  with  the  name  of  the  patient,  the  dilution,  and  the 
time  when  the  preparation  was  made. 

(5)  Place  2  to  5  drops  of  serum  dilution  1 :  20  in  a  second  watch- 
glass,  and  add  an  equal  quantity  of  normal  salt  solution.     Mix  well. 
This  gives  a  dilution  of  1 :  40. 

(6)  Prepare  a  second  slide  by  mixing  a  loopful  of  this  dilution  with 
an  equal  sized  loopful  of  culture.     Mix  gently.     This  gives  a  final  dilu- 
tion of  1 :  80.     Mark  the  slide  with  the  name,  dilution,  and  the  time. 

(7)  Prepare  a  third  slide  by  placing  a  loopful  of  culture  on  a  cover- 
glass  and  invert  over  a  concave  slide  to  which  vaselin  has  been  applied 
in  the  usual  manner.     This  is  the  culture  control.     Label  the  slide. 

(8)  Place  the  slides  in  a  dark  place  at  room  temperature  and  examine 
at  the  end  of  an  hour  with  the  K  or  oil-immersion  lens. 

(a)  First  inspect  the  control.  The  bacilli  should  not  be  clumped, 
but  should  be  motile,  and  preferably  in  the  form  of  long  slender  rods. 
(See  Fig.  81.) 

(6)  Examine  the  1 :  40  and  1 :  80  dilution  preparations:  a  positive 
reaction  is  indicated  by  loss  of  motility  and  definite  clumping  (Fig.  83). 
A  few  free  motile  bacilli  may  be  seen,  or  a  clump  may  be  seen  to  move, 


300 


AGGLUTININS 


owing  to  the  efforts  of  the  bacilli  to  break  away.  A  doubtful  reaction 
is  indicated  by  a  partial  loss  of  motility  and  a  few  indefinite  clumps. 
A  negative  reaction  is  indicated  when  there  is  no  loss  in  motility  or  no 
clumping,  or  when  the  reactions  resemble  the  control  to  which  no  serum 

has  been  added.  In  reporting 
upon  agglutination  tests  always 
state  the  time  at  which  the  test 
was  made  and  the  dilution  used. 

A  1  :  20  and  a  1  :  40  dilution 
may  be  prepared  and  examined  at 
the  end  of  half  an  hour.  Prompt 
agglutination  is  found  practically 
only  in  typhoid  fever. 

Dilutions  may  be  conveniently 
prepared  by  drawing  the  serum  up 
to  the  mark  0.5  in  the  white  cor- 
puscle pipet,  and  the  distilled  water 
up  to  the  mark  11.  Mix  well. 
This  gives  a  dilution  of  1  :  20. 
One  loopful  of  this  diluted  serum 


FIG.  83. — A  POSITIVE  AGGLUTINATION 
(WIDAL)  REACTION  IN  TYPHOID  FE- 
VER. X  430. 

Serum    from   a    patient    ill    about 

twenty-two  days;    a  1  :  100  dilution  at 

the  end  of  one  hour. 


and  one  loopful  of  bouillon  culture 
of  the  microorganism  to  be  tested 

give  a  dilution  of  1  :  40.  One  loopful  of  the  1  :  20  diluted  serum  and 
three  loopful s  of  the  culture  give  a  dilution  of  1 :  80.  Having  mixed  the 
diluted  serum  and  the  bacterial  suspension  on  a  cover-glass,  prepare  the 
cultures  on  the  vaselined  concave  slides  in  the  usual  manner. 


THE  MICROSCOPIC  AGGLUTINATION  TEST  (DRY  METHOD) 

(1)  Place  a  loopful  of  a  twenty-four-hour  bouillon  culture  of  Bacillus 
typhosus  in  the  center  of  a  clean  cover-glass. 

(2)  Moisten  the  dried  blood  which  has  been  collected  on  aluminum 
foil,  glass  slide,  or  paper  with  a  loopful  of  normal  salt  solution.     (A 
second  and  smaller  loop  may  be  used  for  this  purpose.)     Gently  rub  up 
the  dried  blood  and  transfer  a  sufficient  amount  to  the  drop  of  culture 
on  the  cover-glass  until,  when  thoroughly  mixed,  it  presents  a  delicate 
orange  tint  (Fig.  84).     Avoid  transferring  too  much  debris  with  the 
solution  of  blood,  especially  if  the  blood  has  been  collected  on  paper. 
It  is  good  practice  to  mix  the  culture  and  solution  of  blood  with  the 
cover-glass  held  over  a  white  surface,  in  order  that  the  color  may  readily 
be  observed. 


t   *   l*z 


FIG.  84. — MICROSCOPIC  AGGLUTINATION  TEST  WITH  DRIED  BLOOD. 
Shows  the  proper  color  of  the  suspended  drop  of  typhoid  culture  when  the 
solution  of  dried  blood  has  been  added.     The  tinge  should  be  light  orange  or  yellow, 
and  a  shade  lighter  than  ordinary  vaselin  used  in  sealing  the  preparation. 


THE   AGGLUTINATION   REACTION  301 

» .* 

(3)  Having  made  the  mixture  on  the  cover-glass,  invert  it  over  a 
vaselined  concave  slide,  label,  and  stand  aside  for  an  hour. 

(4)  Prepare  the  culture  control  in  the  usual  manner  and  label. 

(5)  Examine  at  the  end  of  an  hour  with  the  y§  or  oil-immersion  lens. 
If  minute  fragments  of  fiber,  etc.,  have  been  transferred,  due  allow- 
ance for  false  agglutination  for  these  should  be  made.     Otherwise  the 
readings  are  made  in  exactly  the  same  manner  as  in  the  "wet"  method. 

(6)  Accurate  dilutions  are  not  possible  with  this  technic.     Satisfac- 
tory results  are  dependent  largely  upon  the  color;    a  faint  orange  tint 
of  the  suspension  is  desirable,  and  probably  represents  a  dilution  of 
about  1 :  40.      This  method,  however,  is  very  simple,  and  when  care- 
fully performed,  yields  results  in  the  practical  serum  diagnosis  of  typhoid 
fever  almost  as  satisfactory  as  the  serum-dilution  method. 

It  is  possible,  however,  to  work  with  known  approximate  dilutions 
by  the  dried  blood  method  if  a  good  chemical  balance  is  available. 
Blood  must  be  collected  on  aluminum  foil  or  glass,  and  is  then  scraped 
off  and  weighed.  To  each  five  milligrams  of  dried  blood  0.5  c.c.  of  salt 
solution  is  added  which  equals  a  dilution  of  1 : 25  of  whole  blood  or 
1 :  100  of  dried  blood  (Wesbrook).  After  permitting  the  mixture  to 
stand  for  half  an  hour  it  is  centrifuged  for  a  short  time.  To  one  drop  of 
the  dilution  thus  obtained  one  drop  of  culture  is  added,  which  gives  a 
final  dilution  of  about  1  :  50.  At  the  end  of  an  hour  it  is  examined. 
Higher  dilutions  can  be  prepared  from  this  stock  dilution  at  the  will  of 
the  operator. 

MACROSCOPIC  AGGLUTINATION  TEST 

This  is  frequently  the  method  of  choice  in  conducting  agglutination 
tests,  especially  in  investigation  and  research  work,  where  a  high  degree 
of  accuracy  is  desirable.  All  dilutions  and  measurements  are  to  be  made 
with  accurate  volumetric  pipets. 

1.  Place  a  row  of  seven  small  test-tubes  (10  x  1  cm.)  in  a  test-tube 
rack  and  add  1  c.c.  of  normal  salt  solution  to  each. 

2.  Dilute  the  serum  1  :  5  in  the  first  tube,  as  follows:  0.2  c.c.  serum 
plus  0.8  c.c.  salt  solution.     This  now  gives  in  this  tube  2  c.c.  of  a  dilution 
of  1 : 10.     Mix  well  with  the  pipet. 

3.  Place  1  c.c.  of  the  serum  from  tube  1  into  tube  2.     Mix  well,  and 
place  1  c.c.  of  the  mixture  from  tube  2  into  tube  3,  and  so  on.     When  the 
sixth  tube  has  been  reached,  discard  1  c.c.,  as  no  serum  is  to  be  added  to 
the  seventh  tube,  which  is  the  culture  control;   i.  e.,  it  will  contain  salt 
solution  plus  bacterial  emulsion. 

4.  Add  1  c.c.  of  bacterial  emulsion  to  each  tube,  which  doubles  the 


302 


AGGLUTININS 


serum  dilution  in  each.  Tube  1  now  contains  a  serum  in  a  dilution  of 
1:20,  acting  on  the  bacteria;  tube  2,  one  of  1:40;  tube  3,  one  of 
1:80;  tube  4,  one  of  1:160;  tube  5,  one  of  1:320;  tube  5,  one  of 
1 : 640.  Tube  7,  as  just  stated,  contains  the  bacterial  emulsion  in  salt 
solution  and  is  the  culture  control, 

In  determining  the  agglutination  titer  of  a  highly  immune  serum, 
these  dilutions  may  be  continued  to  any  degree. 

5.  On  each  tube  the  final  dilution  is  marked  with  a  wax  pencil. 


i 


J 


FIG.  85. — MACROSCOPIC  AGGLUTINATION  REACTION. 

Serum  of  a  person  who  had  received  three  injections  of  typhoid  vaccine.  This 
drawing  was  made  twenty-four  hours  after  the  test  was  set  up.  The  dilutions  are 
marked  on  the  tubes. 

The  tubes  are  then  shaken  gently,  stoppered  with  cotton  plugs,  and 
placed  in  the  incubator  at  37°  C.  or  in  a  water-bath  at  55°  C.  for  two 
hours.  The  tubes  are  then  allowed  to  remain  at  room  temperature  for 
six  hours,  or  in  the  refrigerator  for  twenty-four  hours,  after  which 
readings  are  made. 

6.  The  culture  control  should  show  a  uniform  cloudiness,  with  no 
sediment  or  flakes,  or  at  most  a  very  slight  precipitate  that  is  readily 


^       *  >' 


FIG.  86/ — MACROSCOPIC  AGGLUTINATION  REACTION.     SHOWS  ACTION  OF  AGGLTJTIN- 

OIDS  (PRO-AGGLUTINATION). 

Note  absence  of  agglutination  in  dilution  1 : 50;  agglutination  beginning  in  1 : 100, 
and  fairly  well  marked  to  1:4000  inclusive.  Note  uniform  cloudiness  of  control. 
This  reaction  was  set  up  with  a  typhoid  immune  serum  over  six  months  of  age;  the 
drawing  was  made  after  the  tubes  had  been  incubated  two  hours  and  placed  in  a 
refrigerator  overnight. 

303 


304  AGGLUTININS 

broken  up  by  gentle  agitation.  A  positive  reaction  shows  masses  and 
clumps  of  bacteria  adhering  to  the  sides  and  bottom  of  the  tube,  which 
are  broken  up  with  some  difficulty  (Fig.  85).  The  supernatant  fluid 
should  be  clear.  As  dilutions  become  higher  and  the  amount  of  con- 
tained agglutinin  correspondingly  less,  agglutination  becomes  less  and 
less  complete.  There  is  less  sediment,  and  the  turbidity  of  the  super- 
natant fluid  is  greater,  until  the  negative  tube  closely  resembles  the  cul- 


FIG.  87. — AGGLUTINOSCOPE  (Altaian). 

The  test-tubes  are  arranged  in  the  rack  and  viewed  from  below  in  the  mirror. 
In  this  manner  the  smallest  deposits  are  easily  seen  and  compared  with  the  control. 

ture  control.  A  microscopic  examination  of  a  deposit  will  show  that 
the  bacilli  point  in  all  directions,  whereas  in  a  deposit  of  unagglutinated 
bacilli  they  lie  horizontally  side  by  side. 

When  agglutinoids  are  present,  agglutination  is  absent  or  incomplete 
in  the  lower  dilutions  of  serum,  and  complete  in  the  tubes  containing  the 
higher  dilutions.  This  is  called  pro-agglutination  (Fig.  86). 

Readings  are  facilitated  by  the  use  of  a  special  instrument  known  as 


THE   AGGLUTINATION   REACTION  305 

the  agglutinoscope  (Fig.  87).  The  tubes  are  placed  in  a  rack  having 
numbered  holes,  and  are  viewed  from  beneath  with  the  aid  of  a  mirror. 
In  this  way  one  looks  upward  through  the  column  of  fluid,  and  secures  a 
combined  view  of  sediment  and  turbidity,  and  when  examined  with  the 
culture  control,  fine  and  accurate  readings  may  be  made. 

The  method  of  Kolle  and  Pfeiffer  is  very  convenient,  and  may  be 
safer  than  that  of  adding  live  cultures  with  a  pipet.  It  is  conducted  as 
follows : 

1.  Make  dilutions  of  serum  as  described. 

2.  Emulsify  thoroughly  a  loopful  (2  mg.)  of  culture  from  an  eighteen- 
to  twenty-four-hours-old  agar  culture  in  the  first  test-tube,  repeating 
the  process  in  the  second  tube,  and  so  on  through  the  series.     In  this 
method  the  serum  dilutions  are  not  doubled ;  thus  in  the  foregoing  series 
the  dilutions  would  be  1 :  10,  1 :  20,  1  : 40,  1 : 80,  1 : 160,  1 : 320. 

3.  The  tubes  are  gently  shaken,  labeled,  plugged,  and  incubated  as 
directed  in  the  preceding  method. 

Oxford  University  Method. — This  technic  is  very  serviceable  in 
routine  examinations  in  the  diagnosis  of  typhoid  and  paratyphoid  fever. 

Take  a  stand  containing  15  agglutination  tubes  in  3  rows  of  5  each, 
and  a  dilution  tube. 

With  the  proper  dropping  pipet  measure  out  into  the  dilution  tube 
54  drops  of  normal  saline  solution,  0.85  per  cent,  sodium  chlorid,  in 
distilled  water  (where  the  water  supply  is  pure,  tap-water  can  be  used 
instead  of  saline  solution)  by  means  of  gentle  pressure  on  the  teat. 

Wash  the  pipet  with  distilled  water. 

Dry  out  the  pipet  with  successive  quantities  of  absolute  alcohol, 
followed  by  successive  quantities  of  ether,  and  get  rid  of  the  ether. 

Take  up  the  serum  to  be  tested  into  the  dried  pipet.  Measure  out  6 
drops  of  the  serum  into  the  dilution  tube  already  containing  the  54  drops 
of  saline  solution,  thus  obtaining  a  dilution  of  1  in  10.  Mix  thoroughly. 

Carefully  wash  out  the  pipet. 

With  the  pipet  measure  out  into  each  row  of  tubes  as  follows: 


Drops  of  Drops  of 
Normal  Serum 

Number  Saline  Dilution 

of  Tube.          Solution.  1  in  10. 


0  10 


2 


3  8 

4  9 

5  10 


To  each  tube  in  row  1  add  15  drops  of 
B.  Typhosus  Culture. 

To  each  tube  in  row  2  add  15  drops  of 
B.  Paratyphosus  A.  Culture. 

To  each  tube  in  row  3  add  15  drops  of 
B.  Paratyphosus  B.  Culture. 


20 


306  AGGLUTININS 

At  each  stage  of  the  procedure  the  pipet  is  carefully  washed  and 
dried  out  with  successive  quantities  of  absolute  alcohol  followed  by 
successive  quantities  of  ether. 

Shake  each  tube  thoroughly  in  order  from  right  to  left,  i.  e.,  beginning 
each  row  with  the  highest  dilution. 

Place  the  stain  for  two  hours  in  a  water-bath  at  50°  to  55°  C.  (not 
in  dry  air). 

In  Tube  1  of  each  row  the  serum  acts  in  a  dilution  of  1  in  25. 
In  Tube  2  of  each  row  the  serum  acts  in  a  dilution  of  1  in  50. 
In  Tube  3  of  each  row  the  serum  acts  in  a  dilution  of  1  in  125. 
In  Tube  4  of  each  row  the  serum  acts  in  a  dilution  of  1  in  250. 

Tube  5  containing  no  serum  is  control  against  spontaneous  agglutination. 

If  the  limit  of  agglutination  is  not  reached  within  this  series,  higher 
dilutions  are  followed  out  in  a  similar  manner. 

Thus,  for  example,  57  drops  of  normal  saline  solution  plus  3  drops  of 
a  1  in  10  serum  dilution  will  give  a  serum  dilution  of  1  in  200,  and,  using 
the  same  quantities  as  before,  one  has  the  serum  acting  in  dilutions 
of  1  in  500,  1  in  1000,  1  in  2500,  and  1  in  5000.  And  similarly  for  higher 
dilutions. 

The  tubes  are  examined  after  two  hours  at  50°  to  55°  C.,  followed  by 
fifteen  minutes'  standing  at  room  temperature.  The  reading  is  taken  by 
comparing  each  tube  in  succession  with  the  control  tube,  and  is  pref- 
erably made  by  means  of  artificial  light  against  a  black  background. 
If  daylight  is  used,  the  tubes  inspected  should  be  partly  shadowed  by 
passing  a  finger  up  and  down  behind  them. 

In  non-inoculated  persons  who  have  not  had  typhoid  (or  paratyphoid) 
fever,  agglutination  in  a  dilution  of  1  in  25  justifies  a  strong  suspicion 
of  typhoid  (or  paratyphoid)  infection.  But  the  test  must  be  applied 
again  in  the  course  of  a  few  days  to  ascertain  whether  there  is  any  change 
in  the  titer  of  agglutination.  Marked  agglutination  in  a  dilution  of 
1  in  50  or  more  is  (nearly  always)  diagnostic  of  active  typhoid  (or 
paratyphoid)  infection. 

A  non-inoculated  carrier  will  normally  show  no  important  change 
in  the  titer  of  his  serum  on  repeated  examination  at  short  intervals. 

Inoculated  persons  if  quite  recently  inoculated  will  usually  show  a 
high  titer  of  specific  agglutination.  A  rapid  rise  in  titer  sets  in  within 
two  to  four  days  of  inoculation.  This  is  followed  by  a  fall  at  first  rapid, 
but  subsequently  becoming  very  slow,  so  that  a  relatively  high  titer  is 
maintained  for  a  long  period  (even  for  years) .  During  this  period  ex- 
aminations made  at  intervals  of  a  few  days  give  practically  identical 
readings. 


THE   AGGLUTINATION    REACTION  307 

It  follows  that  in  the  case  of  inoculated  persons  the  diagnosis  of 
active  typhoid  (or  paratyphoid)  infection  will  require  two  or  more 
successive  examinations  of  the  serum. 

(a)  If  the  individual  is  suffering  from  active  typhoid  infection  his 
titer  of  typhoid  agglutination  will  exhibit  the  usual  rise  and  sub- 
sequent regular  fall  seen  in  non-inoculated  subjects,  but  starting 
from  and  returning  toward  the  higher  base  line  of  inoculated 
persons. 

(6)  If  the  individual  is  suffering  from  active  paratyphoid  infection 
one  of  three  things  may  occur  as  regards  his  typhoid  agglutination 
titer,  namely: 

1.  No  appreciable  change  may  occur  in  the  titer  of  typhoid 
agglutination. 

2.  A  relatively  slight  rise  may  occur,  followed  by  a  fall  toward 
the  former  level. 

3.  A  marked  rise  may  occur  synchronous  with  the  rise  in  para- 
typhoid agglutination  titer,  and  subsequently  followed  by 
the  usual  fall  toward  the  former  level. 

Meanwhile  the  titer  of  paratyphoid  agglutination  runs  the  normal 
course  of  rapid  rise  to  a  maximum  (usually  exceeding  the  maximum 
typhoid  titer),  followed  by  a  fall,  at  first  rapid  and  then  slower,  as  already 
described  for  typhoid  subjects,  and  falling  below  the  persistent  base  line 
of  typhoid  agglutination  of  inoculated  persons. 

In  the  case  of  mixed  infections,  whether  in  inoculated  or  non- 
inoculated  persons,  the  agglutinin  curves  for  the  different  infecting 
organisms  are  usually  not  synchronous,  and  they  pursue  their  ordinary 
course  independently  of  each  other. 

Technic  of  Conglutination  Test  with  Bacteria. — Fresh  bovine  serum 
is  heated  to  56°  C.  for  one-half  hour  and  tested  for  agglutinin  for  the 
bacteria  under  study.  If  agglutinin  is  present  it  is  removed  by  adding 
to  each  5  c.c.  of  serum  about  10  loopsful  of  the  corresponding  bacteria, 
mixing  well,  and  removing  the  clumps  by  thorough  centrifuging  and 
filtration  through  paper  after  standing  at  room  temperature  for  several 
hours.  It  may  be  necessary  to  repeat  this  step  once  or  twice  more. 

Dilutions  of  the  patient's  serum  in  amounts  of  1  c.c.  are  made  in 
small  clean  test-tubes,  as  in  the  macroscopic  agglutination  test.  To 
each  tube  add  0.1  c.c.  of  bovine  serum  ("conglutinin") ;  0.1  c.c.  of  fresh 
guinea-pig  serum  (complement)  and  0.1  c.c.  of  the  emulsion  of  the  bac- 
teria of  sufficient  density  to  give  well-marked  emulsions  in  the  test-tubes. 
Controls  of  bovine  serum,  complement  serum,  and  patient's  serum 


308  AGGLUTININS 

should  be  included;  also  a  culture  control  prepared  with  normal  salt 
solution.  After  gentle  mixing  and  standing  at  room  temperature  for 
twenty-four  hours  the  results  are  read;  positive  results  are  indicated 
by  complete  clearing  of  the  tube  with  the  micro-organisms  in  flakes  or 
granules,  either  clinging  to  the  sides  of  the  tube  as  granular  material  or 
at  the  bottom  as  flaky  granular  sediment. 

The  Agglutination  Test  in  the  Differentiation  of  Pneumococci.— 
The  investigations  of  Neufeld  and  Handel,  and  particularly  of  Cole, 
Dochez,  and  their  associates  in  the  Rockefeller  Institute,  have  resulted 
in  the  grouping  of  pneumoccoci  into  four  groups  on  the  basis  of  agglutina- 
tion and  protective  tests.  Group  III  is  composed  of  pneumococci 
belonging  to  the  type  of  pneumococcus  mucosus,  and  an  efficient  anti- 
serum  has  not  yet  been  produced;  Group  IV  is  composed  of  all  pneu- 
mococci not  falling  into  the  first  three  groups. 

Trie  agglutination  test  is  conducted  for  the  purpose  of  determining 
the  presence  of  types  I  or  II;  in  case  either  is  present  and  it  is  decided 
to  administer  an  immune  serum,  the  serum  corresponding  to  the  type 
of  infection  must  be  given.  Either  a  macroscopic  or  microscopic  technic 
may  be  employed;  as  it  is  advisable  to  learn  the  results  as  quickly  as 
possible,  the  latter  saves  time,  although  it  is  generally  considered  as 
being  less  accurate  than  the  former. 

1.  Sputum  coughed  up  from  the  lung  is  collected,  washed  with  sterile 
salt  solution,  and  1  c.c.  injected  into  the  peritoneal  cavity  of  a  mouse. 

2.  If  sputum  is  not  obtainable,  a  culture  may  be  secured  by  puncture 
of  the  lung  after  anesthetizing  the  skin  with  sterile  eucain  solution. 
With  a  sterile  syringe  carrying  2  c.c.  of  warm  sterile  salt  solution  and 
medium-sized  needle  a  puncture  is  made  into  the  consolidated  area  and 
exudate  secured  which  is  injected  into  the  peritoneal  cavity  of  a  mouse. 

3.  Four  to  eight  hours  later  the  mouse  is  killed  with  ether  and  the 
peritoneum  washed  out  with  5  to  10  c.c.  of  normal  salt  solution.     This 
emulsion  is  briefly  centrifuged  to  throw  down  cells  and  the  supernatant 
fluid  transferred  to  a  second  centrifuge  tube  followed  by  rapid  and  thor- 
ough centrifuging  to  throw  down  the  pneumococci.     The  sediment  of 
cocci  is  then  suspended  in  sufficient  normal  salt  solution  to  give  an  even 
emulsion  of  proper  density.     The  supernatant  fluid  maj^  be  used  in 
the  precipitin  test  of  Blake  as  described  on  page  321. 

4.  Macroscopic  tests  are  prepared  by  mixing  in  small  clear  test-tubes 
equal  parts  (0.5  c.c.)  of  bacterial  suspension  and  antipneumococcus  sera 
of  types  I  and  II  undiluted  and  diluted  1  :  5  with  salt  solution.     The 
final  dilutions  are  1  : 2  and  1  :  10.     These  mixtures  are  incubated  at 


THE   AGGLUTINATION   REACTION  309 

37°  C.  for  two  hours,  when  the  results  are  read.  A  control  of  bacterial 
emulsion  and  salt  solution  should  be  included,  and  it  is  advisable  to 
test  out  the  immune  sera  from  time  to  time  with  pure  cultures  of  the 
homologous  strains. 

5.  Microscopic  tests  are  conducted  by  mixing  on  cover-slides  plati- 
num loopsful  of  bacterial  emulsion  and  undiluted  and  diluted  immune 
sera.  These  slides  are  then  suspended  as  hanging-drop  preparations 
and  the  results  read  after  one-half  to  an  hour.  A  control  should  always 
be  included. 

TECHNIC  OF  THE  ABSORPTION  AGGLUTINATION  TEST  IN  MIXED  INFECTION 
(THE  SATURATION  TEST  OF  CASTELLANI) 

The  practical  importance  of  partial  agglutinins  is  recognized  in  the 
diagnosis  of  mixed  infections.  Thus  the  serum  of  a  patient  may  agglu- 
tinate typhoid  as  well  as  paratyphoid  bacilli  in  dilutions  up  to  1 : 100. 
This  may  indicate  one  of  three  possibilities: 

1.  The  patient  may  be  infected  with  typhoid,  but  has  formed  an 
exceptionally  large  quantity  of  group  agglutinins  for  paratyphoid  bacilli. 
Saturation  of  this  serum  with  typhoid  bacilli  will  remove  all  the  typhoid 
and  a  portion,  if  not  all,  of  the  group  agglutinins.     Saturation  with  para- 
typhoid bacilli  will  remove  the  group  agglutinins,  but  not  the  main  or 
typhoid  agglutinin. 

2.  The  patient  may  be  infected  with  paratyphoid  bacilli,  but  has 
formed,  at  the  same  time,  many  partial  agglutinins  for  typhoid  bacilli. 
Saturation  of  the  serum  with  paratyphoid  bacilli  will  remove  all  the 
paratyphoid  and  a  large  portion  of  the  typhoid  agglutinin. 

3.  The  patient  may  have  a  mixed  infection  of  typhoid  and  para- 
typhoid, and  therefore  agglutinin  for  both  may  be  present.     Saturation 
of  the  serum  with  typhoid  bacilli  will  remove  the  typhoid  and  probably 
a  small  portion  of  the  paratyphoid  agglutinin.     After  this  reaction  the 
serum  will  still  show  the  presence  of  a  decided  quantity  of  paratyphoid 
agglutinin. 

In  selecting  the  most  likely  one  of  these  hypotheses  a  decision  may  be 
reached  by  adopting  the  method  of  Castellani  (Citron),  which  is  as  fol- 
lows: 

1.  Four  rows  of  test-tubes  are  arranged,  each  row  being  made  up  of 
four  small  tubes  each  containing  1  c.c.  of  serum  dilutions  1 :  20,  1 : 40, 
1  :  80,  and  1 : 160  respectively. 

2.  In  each  of  the  tubes  of  the  first  and  second  rows  five  loopfuls  of 
typhoid  bacilli  are  emulsified.     An  extra  tube  containing  1  c.c.  of  nor- 


310 


AGGLUTININS 


mal  salt  solution  receives  a  similar  amount  of  bacteria,  and  serves  as  the 
typhoid  control. 

3.  In  each  tube  of  the  third  and  fourth  rows  five  loopfuls  of  para- 
typhoid bacilli  are  emulsified.     Arrange  the  paratyphoid  culture  control. 

4.  Mix  gently  and  incubate  for  four  hours.     Carefully  record  the 
presence  or  absence  of  agglutination  in  each  test-tube.     Centrifuge  all 
the  tubes  excepting  the  two  controls,  and  transfer  the  supernatant  fluid 
of  each  to  other  test-tubes  arranged  in  the  same  order. 

5.  To  each  tube  of  the  first  and  third  rows  add  five  loopfuls  of  ty- 
phoid bacilli;    to  each  of  the  second  and  fourth  rows,  five  loopfuls  of 
paratyphoid  bacilli.     Mix  well  and  incubate  for  four  hours. 

(a)  If  typhoid  is  present,  the  agglutination  titer  in  the  first  part  of 
the  test  will  be  strong  in  the  tubes  of  the  first  and  second  rows,  and  weak 
in  those  of  the  second  and  third  rows.  In  the  second  part  of  the  test  the 
titer  for  typhoid  will  be  weak  or  nil  in  the  first,  second,  and  fourth  rows, 
whereas  in  the  third  row  it  will  remain  practically  the  same. 

(6)  If  paratyphoid  exists,  the  agglutination  titer  in  the  first  part  of 
the  test  will  be  strong  in  the  tubes  of  the  third  and  fourth  rows,  and  weak 
in  those  of  the  first  and  second  rows.  In  the  second  part  of  the  test  the 
titer  for  paratyphoid  will  be  less  or  nil  in  the  fourth  row,  and  strong  or 
unchanged  in  the  second  row. 

(c)  If  a  mixed  infection  exists,  the  agglutination  titer  in  the  first  part 
of  the  test  will  be  strong  in  the  tubes  of  all  four  rows.  In  the  second 
part  of  the  test  the  titer  in  the  first  and  fourth  rows  is  much  weaker  or 
nil,  and  in  the  second  and  third  rows  it  will  remain  the  same. 

Technic  of  Acid  Agglutination. — Mixtures  of  lactic  acid  and  sodium 
lactate  have  been  generally  employed.  Gillespie  has  prepared  these  from 
stock  solutions  of  one-third  normal  sodium  lactate,  with  a  small  crystal 
of  thymol,  and  normal  lactic  acid,  according  to  the  following  scheme: 


N 

—  sodium  lactate  in  c.e 

3 

Normal  lactic  acid  in  c.c 

Distilled  water  in  c.c 

Ratio  of  acid  to  salt 

H  ion  concentration  in  grams  per 
multiplied  by  104 


1.5 


0.12  0.25 

8 
18.4 

0.04 


1.5    1.5 

0.5 


18.2 


0.1 


0.2 


1.5    1.5 
1.0   2.0 


8 
18.017.516.5 

I 


0.4    0.7 


1.5 

0.5 


1.5 


1.0 


18.0  17.5 
1 

1.4   2.8 


1.5 


2.0 


5.5 


1.5 

4.0 


16.5  14.5 


11 


1.5 


8.0  16.0 


10.5 
16 

22 


1.5 


2.5 
32 


44 


A  series  of  greater  dilutions  of  lactates  may  be  made  by  diluting 
each  of  the  above  with  one  or  more  volumes  of  distilled  water. 


1  Read  0.12  c.c.  of  normal  acid  freshly  diluted  1  : 8. 


THE    AGGLUTINATION    REACTION  311 

Young  cultures  of  bacteria  should  be  employed;  if  grown  in  broth, 
they  may  be  prepared  by  rapid  centrifuging  and  resuspension  in 
sufficient  distilled  water  to  secure  an  even  emulsion  of  such  density 
as  suitable  for  the  macroscopic  agglutination  test — 0.3  c.c.  of  the 
bacterial  emulsion  is  placed  in  each  of  a  series  of  small  and  perfectly 
clean  tubes  in  a  rack,  and  1  c.c.  of  the  proper  reaction  mixture  added 
to  each  tube.  The  tubes  are  then  gently  and  briefly  shaken  and  placed 
in  a  water-bath  at  about  37.5°C.  for  an  hour  or  two.  Michalis1  uses 
acetic  acid  and  reports  sharp  differentiation  of  Bacillus  typhosus  by 
the. method  of  acid  agglutination. 

TESTS  BEFORE  TRANSFUSION  FOR  ISOHEMAGGLUTININS 
AND  ISOHEMOLYSINS 

1.  Two  or  three  c.c.  of  blood  are  obtained  from  each  donor  from  a 
vein  at  the  elbow  and  0.5  c.c.  is  placed  at  once  in  a  centrifuge  tube  con- 
taining 5  c.c.  of  a  1  per  cent,  sodium  citrate  in  normal  salt  solution. 
The  remainder  is  placed  in  a  small,  dry  test-tube  until  coagulation  has 
occurred  and  the  serum  has  separated. 

2.  From  the  recipient,  3  to  4  c.c.  of  blood  are  necessary;   0.5  c.c.  is 
placed  in  sodium  citrate  solution,  and  the  remainder  is  allowed  to  coagu- 
late and  the  serum  collected. 

3.  The  sodium  citrate  tubes  are  centrifuged;  the  supernatant  fluid 
is  pipeted  off,  and  the  cells  are  washed  again  with  normal  salt  solution. 
After  the  final  washing  enough  normal  salt  solution  is  added  to  the  sedi- 
ment of  cells  to  bring  the  total  volume  up  to  5  c.c. 

4.  The  serum  tubes  are  also  centrifuged,  so  that  clear  serums  are 
obtained.     These  should  preferably  be  free  from  hemoglobin  stain. 

5.  The  following  mixtures  should  be  set  up  within  twenty-four  hours 
of  the  time  of  collecting  blood,  in  order  that  native  complements  may 
not  have   undergone   deterioration.     Measurement  may  be  made  ac- 
cording to  a  drop  from  an  ordinary  1  c.c.  graduated  pipet  held  vertically. 
Small  sterile  test-tubes  (8  by  1  cm.)  are  to  be  used. 

Tube  1 :  4  drops  of  donor's  serum  +  1  drop  of  recipient's  red-cell 

emulsion. 
Tube  2 :  4  drops  of  recipient's  serum  +  1  drop  of  donor's  red-cell 

emulsion. 
Tube  3:   Control:   4  drops  of  donor's  serum  +  1  drop  of  donor's 

red-cell  emulsion.     Should  show  no  agglutination  and  no  hem- 

olysis. 

1  Deutsch.  Med.  Wchnschr.,  1915,  xli,  241. 


312  AGGLUTININS 

Tube  4:  Control:  4  drops  of  recipient's  serum  +  1  drop  of  re- 
cipient's red-cell  emulsion.  Should  show  no  agglutination  or 
hemolysis. 

Tube  5:    Control:    1  drop  of  donor's  red-cell  emulsion  -J-  4  drops 
of  normal  salt  solution.     This  serves  as  a  control  on  the  toxicity 
of  the  corpuscles  and  isotonicity  of  the  salt  solution. 
Tube  6:    Control:    1   drop  of  recipient's  red-cell  emulsion  +  4 

drops  of  saline  solution. 

One  cubic  centimeter  of  salt  solution  is  added  to  each  tube  and  the 
tubes  are  gently  shaken  and  placed  in  the  incubator  for  two  hours. 
They  are  to  be  inspected  every  half  hour.  Agglutination  is  recognized 
macroscopically  by  the  clumping  of  the  red  blood-cells  into  small  masses 
that  later  sink  to  the  bottom  of  the  tube  as  a  small  clot.  Hemolysis 
is  likewise  easily  detected,  as  corpuscles  tend  to  become  precipitated 
within  two  hours.  If  doubt  exists,  the  finer  grades  of  hemolysis  may  be 
detected  after  the  tubes  have  been  allowed  to  stand  over  night  in  an  ice- 
chest,  or  at  once  by  thorough  centrifugalization. 

Pipet  Method. — In  cases  where,  for  any  reason,  the  quantities  of 
blood  previously  named  cannot  be  secured,  the  whole  operation  may  be 
conducted  with  smaller  amounts,  using  Wright's  capillary  pipets 
(Epstein  and  Ottenburg).  This  method  is  as  follows: 

Blood  is  secured  from  both  recipient  and  donor  by  pricking  the  finger 
of  each  and  allowing  five  or  six  drops  of  blood  to  flow  into  5  c.c.  of  sodium 
citrate  solution,  and  collecting  a  good-sized  Wright  capsule  full  for  se- 
curing the  serum.  The  corpuscles  are  washed  twice  and  enough  normal 
salt  solution  added  to  make  up  approximately  a  10  per  cent,  suspension. 
The  capsules  are  centrifuged  if  necessary,  filed,  opened,  and  the  serum 
pipeted  off. 

The  mixtures  are  prepared  in  Wright's  capillary  pipets  (see  Fig.  48) 
fitted  with  rubber  nipples.  The  unit  volume  is  marked  off  by  a  blue 
pencil  on  the  stem  about  an  inch  from  the  tip.  Four  volumes  of  serum, 
one  volume  of  cell  emulsion,  and  four  or  five  volumes  of  salt  solution 
are  drawn  up  into  the  pipet  and  then  mixed  gently,  running  them  out  on 
a  watch-glass.  The  entire  mixture  is  then  drawn  into  the  barrel  of  the 
pipet  and  the  tip  sealed.  The  pipets  are  carefully  labeled,  incubated  for 
two  hours,  and  examined  for  agglutination  and  hemolysis. 

The  results  are  somewhat  more  difficult  to  read,  but  the  method  is 
of  value  when  many  donors  are  to  be  examined  or  when  the  supplies  of 
serum  and  corpuscles  are  limited. 

Microscopic  Hemagglutination  Method  of  Brem.1 — The  following 
1  Jour.  Amer.  Med.  Assoc.,  1916,  67,  191. 


TESTS    FOR   ISOHEMAGGLUTININS   AND    ISOHEMOLYSINS  313 

technic  is  especially  recommended  for  rapidity  and  grouping;  the  masking 
of  agglutination  by  hemolvsis  is  delayed  or  prevented.  This  method  of 
grouping  should  be  used  in  conjunction  with  the  group  of  Moss  as  given 
on  page  286.  It  is  necessary  to  have  on  hand  a  Group  2  or  Group  3 
blood,  and  the  laboratory  worker  should  know  the  grouping  of  his  own 
blood  to  be  used  in  this  work.  Group  2  or  Group  3  serum  may  be  pre- 
served in  a  sterile  condition  in  the  refrigerator  for  several  months 
without  losing  agglutinating  properties.  Group  2  and  Group  3  corpus- 
cles may  be  preserved  for  four  weeks  in  the  refrigerator  by  the  method 
of  Wohl,1  who  adds  3  drops  of  blood  to  each  cubic  centimeter  of  a  solu- 
tion prepared  by  adding  0.5  c.c.  of  40  per  cent,  formaldehyd  solution 
to  500  c.c.  of  0.85  per  cent,  sodium  chlorid  solution  containing  2  per 
cent,  sodium  citrate. 

Five  or  6  drops  of  Group  2  blood  are  collected  in  a  small  clean,  dry 
test-tube  or  centrifuge  tube,  and  2  drops  in  another  tube  containing 
1  c.c.  of  1.5  per  cent,  sodium  citrate  in  0.9  per  cent,  salt  solution.  The 
serum  and  corpuscles  of  the  unknown  blood  to  be  grouped  are  collected 
in  the  same  manner.  The  bloods  in  the  dry  tubes  are  allowed  to  coagu- 
late, the  coagula  loosened  with  a  platinum  loop,  and  the  sera  separated 
by  centrifuging.  Sera  and  corpuscles  are  now  ready  for  the  tests.  Two 
platinum  loopsful  of  serum  are  mixed  with  one  loopful  of  corpuscle 
suspension  on  a  cover-slide  and  inverted  over  an  ordinary  hanging- 
drop  slide  ringed  with  vaselin.  Each  serum  is  set  up  with  each  corpuscle 
suspension.  Agglutination,  if  it  occurs,  takes  place  at  room  temperature 
within  five  minutes;  the  slides  should  be  examined  at  once  and  at  short 
intervals  with  a  f  objective  of  the  microscope,  and  rouleaux  formation 
must  be  differentiated  from  small  clumps  due  to  agglutination. 

1  Jour,  of  Lab.  and  Clin.  Med.,  1917,  2,  516. 


CHAPTER  XVII 
PRECIPITINS 

CLOSELY  allied  to  the  agglutinins  are  antibodies  known  as  precipitins. 
They  act  on  dissolved  albuminous  bodies  in  a  manner  quite  similar  to 
the  action  of  agglutinins  upon  formed  cellular  elements.  For  example: 
(1)  If  typhoid  immune  serum  is  added  to  a  bouillon  culture  of  typhoid 
bacilli,  agglutination  occurs;  (2)  if  the  culture  is  filtered  and  the  im- 
mune serum  is  added  to  the  clear  sterile  filtrate,  cloudiness  appears  and 
finally  a  precipitate  forms.  The  first  is  an  example  of  the  action  of 
agglutinins  upon  the  formed  bacilli,  and  the  second  illustrates  the  ac- 
tion of  precipitins  upon  the  albumins  of  dead  and  dissolved  bacilli. 

Precipitins  are  formed  not  only  for  bacterial  albumins,  but  for  most 
any  soluble  animal  (zooprecipitin)  and  vegetable  protein  (phytoprecipi- 
tiri)  as  well.  If  the  serum  of  a  rabbit  immunized  with  horse  serum  is 
added  to  horse  serum,  a  precipitate  forms,  owing  to  the  presence  of  a 
specific  precipitin  in  the  immune  serum.  Normal  rabbit  serum  does  not 
possess  this  power. 

Definition. — The  precipitins  are  specific  antibodies  that  develop  in  the 
serum  of  animals  inoculated  with  bacteria  or  with  solutions  of  animal  or 
vegetable  albumins,  which  possess  the  power  of  producing  a  precipitate  in  a 
.  dear  solution  of  the  particular  albumin  or  culture  filtrate  against  which  the 
animal  has  been  immunized. 

Historic. — Kraus  (1897)  was  the  first  to  study  and  describe  the  bac- 
terial precipitins.  He  observed  that  when  the  serums  of  animals  that 
have  been  immunized  against  cholera,  typhoid,  or  plague  are  added  to  a 
clear  filtrate  of  the  respective  bouillon  cultures  of  their  bacteria,  instead 
of  to  the  bacteria  themselves,  the  clear  solution  becomes  turbid  and  a 
precipitate  forms. 

This  reaction  was  found  to  be  quite  specific,  i.  e.,  it  occurs  best  with 
the  filtrate  of  the  homologous  bacteria,  and  to  a  much  less  extent  with 
closely  allied  species.  For  example,  the  typhoid  immune  serum  does 
not  produce  a  precipitate  with  a  filtrate  of  Spirillum  cholerse,  and  simi- 
larly a  cholera  immune  serum  does  not  produce  a  precipitate  with  the 
filtrate  of  Bacillus  typhosus.  Kraus  advocated  the  precipitin  reaction 

314 


HISTORIC  315 

as  a  means  of  identifying  and  differentiating  certain  species  of  bacteria, 
but  the  test  possesses  no  advantage  for  these  purposes  over  agglutina- 
tion reactions  and  is  not  generally  employed. 

Tchistovitch  was  the  first  to  call  attention  to  the  non-bacterial  pre- 
cipitins.  This  observer  found  that  the  serum  of  rabbits  inoculated  with 
eel  serum,  when  mixed  with  a  small  quantity  of  the  eel  serum,  caused  a 
precipitate  to  form. 

About  the  same  time  (1899)  Bordet  found  that  the  serum  of  rabbits 
inoculated  with  the  serum  of  chickens,  when  mixed  with  the  chicken 
serum,  gave  a  specific  precipitate.  A  little  later  Bordet  produced  an 
anti-milk  immune  serum  (lactoserum)  by  inoculating  rabbits  intraperi- 
toneally  with  milk  partially  sterilized  by  heating  to  65°  C.  When  this 
immune  serum  was  mixed  with  the  homologous  milk,  small  particles 
appeared,  which  gradually  formed  larger  flak.es  and  sank  to  the  bottom 
of  the  fluid.  It  was  found  that  the  lactoserums  were  specific — i.  e., 
cow  lactoserum  would  precipitate  only  cow  casein,  human  serum  only 
human  casein,  etc. 

Wladimiroff  was  the  first  to  use  the  bacterial  precipitin  reaction  as  a 
practical  diagnostic  test.  He  showed  that  the  serum  of  a  horse  suffering 
from  glanders  would,  when  added  to  a  clear  filtrate  of  a  culture  of  Bacil- 
lus mallei,  produce  a  precipitate.  The  technic  of  these  reactions  is, 
however,  more  difficult  than  with  the  agglutination  tests,  and  as  the 
reactions  are  usually  not  more  delicate  or  more  advantageous  than  the 
latter,  they  are  seldom  employed. 

Following  Wladimiroff,  Uhlenhuth  and  Wassermann  made  a  very 
important  practical  demonstration  of  the  value  of  serum  precipitins  in 
differentiating  the  blood  and  secretions  of  man  and  animals.  For  ex- 
ample, the  serum  of  rabbits  immunized  with  various  bloods  would  react 
with  solutions  of  old  and  dried  specimens  of  their  respective  bloods,  and 
although  " group"  precipitins  were  found  present  in  the  tests  with  the 
blood  of  closely  allied  species,  yet  the  value  of  the  reaction  was  not 
impaired  to  any  extent  when  a  proper  technic,  with  correct  dilutions,  was 
employed.  These  discoveries  were  found  to  possess  considerable  value 
in  forensic  medicine,  particularly  in  the  recognition  of  the  source  of 
blood-stains. 

Nuttall,  in  a  thorough  and  painstaking  research  with  the  blood  from 
500  animals,  was  able  to  study  the  "blood  relationship"  of  various  ani- 
mals as  based  upon  group  precipitins.  For  example,  the  serum  of  a 
rabbit  immunized  with  human  blood  will  react  best  with  human  serum, 
then  with  the  serums  of  the  higher  apes,  and  finally  with  the  lower  orders 


316  PRECIPITINS 

of  monkeys.  Similar  reactions  were  found  to  occur  among  the  lower 
animals. 

Nomenclature. — The  antibody  in  an  immune  serum  responsible  for 
the  phenomenon  of  precipitation  is  called  precipitin;  the  substance  or 
antigen  responsible  for  the  production  of  this  antibody  is  known  as  the 
precipitinogen;  the  precipitate  is  the  end-product  of  the  reaction  between 
precipitinogen  and  precipitin.  Just  as  toxoids  and  agglutinoids  may  be 
formed,  so  precipitin  may  be  modified  to  precipitoid. 

Although,  since  bacterial  precipitins  are  produced  by  the  protein 
constituents  of  bacteria,  the  custom  of  differentiating  between  bacterial 
and  protein  precipitins  is  superfluous,  nevertheless,  when  the  mean- 
ing is  clearly  understood,  the  term  bacterial  precipitin  is  convenient 
and  may  be  employed. 

The  precipitins  derive  their  names  from  their  precipitinogens,  as, 
for  example,  a  precipitin  produced  by  injecting  rabbits  with  ox  serum 
is  designated  anti-ox  precipitin. 

Normal  Precipitins. — Although  agglutinins  may  be  found  in  normal 
serum,  it  is  decidedly  uncommon  to  find  normal  precipitins.  Extracts 
of  organs  have  been  known  to  contain  normal  precipitins  for  certain 
albumins,  although  at  the  same  time  they  were  absent  from  the  serum 
of  the  animal.  In  this  case  the  active  bodies  exist  in  the  cells  as  "ses- 
sile receptors,"  and  by  the  process  cf  extraction  they  are  brought  into 
solution.  During  immunization  these  same  receptors  are  stimulated  to 
overproduction  and  are  thrown  into  the  circulation  as  free  precipitin 
receptors. 

Immune  precipitins  are  antibodies  produced  by  immunization  with 
a  foreign  albumin,  either  during  the  course  of  a  bacterial  infection  or  as 
the  result  of  artificial  inoculation. 

Structure  and  Properties  of  Precipitins. — According  to  the  side-chain 
theory,  precipitins  are  antibodies  or  receptors  of  the  second  order,  com- 
posed of  a  combining  arm  or  haptophore  group  for  the  precipitinogen, 
and  a  zymophore  or  precipitinophore  group  that  precipitates  the  antigen. 
Their  structure  is,  therefore,  seen  to  be  quite  similar  to  that  of  agglu- 
tinin,  the  difference  being  largely  due  to  the  different  functions  of  the 
zymophore  group. 

The  properties  of  precipitins  are  quite  similar  to  those  of  agglutinins. 
They  are  fairly  resistant  bodies,  resist  the  effect  of  drying  for  prolonged 
periods,  but  are  gradually  destroyed  by  heating  to  60°  to  70°  C.  When 
inactivated  by  exposure  or  heat,  they  cannot  be  reactivated  by  the  addi- 
tion of  fresh  normal  serum,  and  therefore  they  bear  no  relation  to  the 


FORMATION   OF   PRECIPITINS  317 

complements.  As  with  agglutinins,  the  presence  of  a  salt  is  necessary 
to  secure  the  reaction. 

The  haptophore  or  combining  arm  is  quite  stable;  the  precipito- 
phore  group  is  more  labile,  and  is  affected  by  heat,  and  when  this  less 
resistant  arm  is  lost,  the  receptor  is  called  a  precipitoid.  Like  agglutin- 
oids,  the  precipitoids  are  of  practical  interest  from  the  fact  that  their 
haptophore  arm  will  not  only  combine  with  precipitinogen,  but  displays 
a  greater  activity  in  this  direction  than  the  whole  receptor  or  precipi- 
tin  itself,  and  when  union  between  precipitinogen  and  precipitoid  has 
occurred,  precipitation  does  not  result.  Hence  in  low  dilutions  of  a 
precipitin  serum  the  phenomenon  of  precipitation  is  slight  or  altogether 
absent,  whereas  in  higher  dilutions  the  reaction  becomes  evident. 

Group  precipitins  are  not  so  prominent  as  group  agglutinins,  yet  they 
are  formed  to  a  certain  degree  and  are  of  much  practical  importance  in 
attempting  to  differentiate  bacteria  and  serums  by  the  precipitation 
method.  Although  precipitins  are  highly  specific,  the  principle  of  serum 
dilution,  as  emphasized  under  Agglutination,  must  be  closely  observed 
in  order  to  dilute  the  group  precipitins  to  such  small  amounts  as  to  pre- 
vent them  from  interfering  with  the  chief  precipitin.  This  principle  is  of 
particular  importance  in  differentiating  the  bloods  of  various  animals, 
and  especially  in  medicolegal  cases,  where  the  precipitin  reactions  are 
employed  for  the  diagnosis  of  blood-stains. 

Formation  of  Precipitins. — Immune  serums  for  diagnostic  purposes 
are  produced  by  injecting  the  precipitinogenous  fluid  into  the  veins, 
peritoneal  cavity,  or  subcutaneous  tissues  of  animals,  usually  rabbits. 
The  power  of  forming  precipitins  is  probably  disseminated  among  the 
organs  and  general  body  tissues.  Kraus  and  Levaditi  assign  the  leu- 
kocytes as  the  chief  source  of  precipitin  formation. 

As  in  the  case  of  agglutinin  formation,  not  all  animals  possess  equally 
the  power  of  forming  a  precipitin  for  a  given  albumin.  While  this  point 
is  of  general  interest  with  the  bacterioprecipitins,  it  becomes  of  particular 
importance  in  relation  to  serum  precipitins.  For  example,  an  animal 
will  not  form  a  precipitin  active  against  its  own  serum.  If  formed,  it 
would  be  an  autoprecipitin,  or  isoprecipitin,  and,  as  a  rule,  animals  do 
not  form  antibodies  for  their  own  tissue  constituents.  Furthermore, 
animals  are  unlikely  to  form  precipitins  for  the  proteins  of  other  members 
of  the  same  species,  or  if  precipitins  are  produced,  they  are  usually  the 
result  of  prolonged  immunization  of  a  number  of  animals.  Precipitin 
formation  is  also  slight  for  the  proteins  of  other  animals  that  are  closely 
related  either  zoologically  or  biologically.  For  example,  attempts  at 


318  PRECIPITINS 

immunization  of  a  guinea-pig  with  the  serum  of  a  rabbit,  a  pigeon  with 
that  of  a  hen,  or  a  monkey  with  human  serum,  are  procedures  that  do  not 
usually  yield  good  precipitating  serums. 

Attempts  have  been  made  to  produce  antiprecipitin  by  effecting 
immunization  with  immune  precipitating  serums.  Such  attempts  have 
been  reported  as  partially  successful  with  serum  and  milk,  but  not  with 
bacterial  precipitins.  Antiprecipitins  possess  no  practical  value. 

Mechanism  of  Precipitation. — Of  the  various  theories  advanced  to 
explain  the  phenomenon  of  precipitation,  none  has  received  so  much 
support  experimentally  as  that  advanced  by  Bordet  in  explanation  of 
agglutination. 

Colloids  may  be  precipitated  by  salts,  and  probably  the  salts  so  alter 
the  electric  state  of  colloidal  particles  that  their  surface  tension  is  de- 
creased, and,  as  a  result  of  this. change,  neighboring  particles  coalesce 
in  such  quantities  as  to  produce  a  visible  precipitate.  Salts  are  likewise 
necessary  for  serum  precipitation,  and  there  is  a  close  analogy  between 
serum  and  colloidal  precipitation. 

The  origin  of  the  precipitate  formed  during  the  reaction  is  of  interest. 
When  a  very  potent  immune  serum  is  employed,  the  precipitinogen  is  so 
highly  diluted  that  it  no  longer  gives  any  of  the  chemical  reactions  for 
proteins,  but  when  the  precipitating  serum  is  added,  it  may  yield,  never- 
theless, a  heavy  precipitate.  The  precipitate  can,  therefore,  hardly  be 
regarded  as  due  to  the  slight  trace  of  albumin  in  the  precipitinogen,  and, 
furthermore,  if  the  precipitating  serum  is  diluted,  the  precipitate  be- 
comes smaller  and  smaller,  and,  if  the  dilution  is  increased,  it  finally 
disappears  altogether.  For  this  reason  the  precipitate  is  generally 
considered  as  originating  in  the  immune  serum. 

Specificity  of  Precipitins. — Precipitins  react  but  feebly  on  closely 
related  albumins  of  the  same  species,  but  are  specific  against  those  of 
unrelated  species.  In  other  words,  the  precipitation  test  merely  deter- 
mines the  animal  species  from  which  the  proteid  originates,  but  cannot 
demonstrate  positively  whether  it  comes  from  the  blood,  the  semen, 
milk,  or  other  albuminous  body.  For  medicolegal  purposes,  therefore, 
a  diagnosis  of  "human  blood-stain"  cannot  be  made  without  chemical 
evidence  to  prove  that  the  stain  actually  consists  of  blood. 

An  immune  serum  prepared  by  the  injection  of  the  serum  of  a  certain 
animal  gives  a  precipitate  also  with  the  juices  of  the  various  organs  of 
that  animal.  The  only  exception  to  this  rule  is  the  protein  of  the  crystal- 
line lens  of  the  eye,  which  gives  no  precipitate  with  the  antiblood  im- 
mune serum.  The  same  albumin  exists  in  the  crystalline  lenses  of  all 


R6LE    OF   PRECIPITINS    IN    IMMUNITY  319 

animals, — from  fishes  to  man, — and  a  serum  produced  by  immunization 
with  lens  substance  will  react  with  the  protein  derived  from  the  lens  of 
any  animal,  but  with  no  other  animal  proteid.  This  theory  of  species 
specificity  may,  however,  be  carried  a  little  further,  for  by  'carefully 
freeing  the  organs  from  all  blood  and  then  using  organic  extracts  for 
inoculation,  it  is  possible  to  produce  serums  that  will  yield  a  precipitate 
best  with  the  particular  variety  of  organic  extract  (liver,  kidney,  etc.), 
and  a  weak  or  no  precipitate  with  extracts  of  other  organs  of  the  same 
animal. 

Besides  this  animal  specificity,  precipitin  reactions  also  demonstrate 
the  "constitutional  specificity"  of  proteins.  If,  instead  of  using  a  pure 
animal  or  plant  albumin  for  immunization,  variously  altered  albumins 
are  used  (heated  albumins,  acid  albumin,  formaldehyd  albumin,  and  the 
like),  the  organism  reacts  by  producing  antibodies  of  a  characteristic 
nature,  differing  from  those  developed  after  inoculation  with  pure  al- 
bumin. For  example,  if  a  rabbit  is  immunized  with  normal  horse  serum, 
the  resulting  immune  serum  will  produce  a  precipitate  when  added  to 
pure  horse  serum,  but  not  when  added  to  horse  serum  that  has  been 
heated.  On  the  other  hand,  if  a  rabbit  is  inoculated  with  horse  serum 
that  has  been  diluted  and  boiled  for  a  short  time,  the  resulting  immune 
serum  will  react  not  only  with  normal  horse  serum,  but  also  with  heated 
serum  and  a  group  of  its  decomposition  products  with  which  the  normal 
immune  serum  ordinarily  never  produces  a  precipitate. 

This  observation  is  of  practical  importance  in  detecting  meat  sub- 
stitution by  precipitin  reactions.  In  order  to  render  the  detection  dif- 
ficult, the  meat  is  commonly  boiled ;  with  the  aid  of  precipitins  produced 
by  immunization  with  heated  proteins,  this  fraud  is  more  easily  detected 
than  if  a  normal  immune  .serum  were  used. 

Obermeyer  and  Pick  have  demonstrated  that  while  animal  specificity 
is  not  destroyed  when  the  albumins  are  modified  by  heat,  tryptic  di- 
gestion, or  oxidation,  their  specificity  is  lost  when  an  iodin,  nitro-  or 
diazo-group  is  inserted  into  the  protein  molecule.  Immunization  with 
such  transformed  proteins,  e.  g.,  xanthoprotein,  can  produce  a  precipi- 
tating serum  that  will  react  with  every  xanthoprotein,  even  that  of 
different  animals.  These  investigators  conclude  that  species  specificity 
is  probably  dependent  upon  a  certain  aromatic  group  of  the  protein 
molecule. 

Role  of  Precipitins  in  Immunity. — Precipitins  are  probably  not  truly 
protective  antibodies,  like  antitoxin  and  the  lysins,  but  they  are  quite 
similar  to  the  agglutinins  in  being  secondary  products  of  cellular  activ- 


320  PRECIPITINS 

ity,  and  they  are  of  value  chiefly  as  indicators  of  this  general  antibody 
formation.  They  may,  however,  be  concerned  in  preparing  their  anti- 
gens for  destruction  and  solution,  just  as  opsonins  prepare  cells  for  pha- 
gocytosis, but  precipitins  themselves  possess  no  appreciable  curative  or 
protective  virtues,  and  are  of  value  chiefly  in  diagnostic  procedures. 


PRACTICAL  APPLICATIONS 
BACTERIAL  PRECIPITINS 

Bacterial  precipitins  have  no  clinical  diagnostic  value.  Their  re- 
actions have  no  advantage  over  the  agglutination  test,  they  are  more 
difficult  of  execution  than  the  latter,  the  sources  of  error  are  greater. 

In  scientific  research  they  may  be  of  value  in  differentiating  micro- 
organisms from  closely  allied  species,  but  even  here  agglutination 
reactions  serve  the  purpose  equally  well  and  are  less  difficult  of 
execution. 

Occasionally  bacterial  precipitins  are  of  service  in  demonstrating 
the  presence  of  soluble  bacterial  substances  within  exudates  or  organic 
fluids.  For  example,  Vincent  and  Bellot  recommend  the  reaction  as 
being  of  considerable  value  in  the  diagnosis  of  cerebrospinal  meningitis. 
The  cerebrospinal  fluid  is  centrifugalized  until  it  is  clear;  2  c.c.  of  this 
clear  fluid  is  then  placed  in  one  tube  and  4  c.c.  into  another.  One-tenth 
cubic  centimeter  of  a  standard  antimeningococcic  serum  is  added  to 
each  tube.  The  tubes  are  kept  for  from  twelve  to  fifteen  hours  at  37°  C. 
In  the  presence  of  cerebrospinal  meningitis  a  precipitate  forms.  If  the 
fluid  is  normal  or  if  it  was  derived  from  some  other  form  of  meningitis, 
no  cloudiness  results.  This  reaction  is  said  to  occur  within  the  first 
twenty-four  hours  of  the  illness  and  to  persist  until  the  twelfth  to  the 
twentieth  day. 

Similar  reactions  have  been  advocated  in  the  diagnosis  of  other  in- 
fections, particularly  syphilis.  Fornet  believed  that  the  presence  of 
typhoid  antigen  (precipitinogen)  ought  to  be  capable  of  demonstration 
in  the  blood-serum  of  typhoid  fever  patients  long  before  antibodies 
themselves  are  in  evidence.  One  cubic  centimeter  of  potent  immune 
serum  in  concentrated  and  diluted  form  (1:5  and  1 : 10  with  normal  salt 
solution)  is  placed  into  small  test-tubes,  and  an  equal  amount  of  the 
serum  for  examination,  also  in  concentrated  and  similar  dilutions,  is 
carefully  floated  on  top  of  the  immune  serum.  Control  tests  with  nor- 
mal and  immune  serum  and  normal  with  unknown  serum  are  made. 
The  mixtures  are  allowed  to  stand  undisturbed  at  room  temperature  for 


PRACTICAL   APPLICATIONS  321 

two  hours,  and  if  the  reaction  is  positive,  a  whitish  ring  makes  its  appear- 
ance at  the  point  of  contact  of  the  two  serums,  the  controls  remaining 
negative.  According  to  Citron,  this  ring  test  is  also  evident  in  the  pres- 
ence of  scarlet  fever,  measles,  and  syphilis. 

Precipitin  Reaction  in  Pneumonia. — Dochez  and  Avery  have  demon- 
strated the  presence  of  a  soluble  pneumococcus  substance  specific  for 
the  type  strain  in  the  blood  and  urine  of  persons  suffereng  with  pneu- 
monia, and  found  a  precipitin  reaction  of  value  in  the  diagnosis  of  the 
type  of  pneumococcus  causing  the  infection.  Equal  parts  (0.5  to  1.0  c.c.) 
of  fresh  clear  serum  or  urine  are  mixed  in  small  clean  test-tubes  with 
antipneumococcus  sera  I  and  II;  a  distinct  cloud  which  becomes  more 
definite  after  standing  an  hour  develops  with  the  immune  serum  corre- 
sponding to  the  type  strain  of  infection  if  due  to  types  I  or  II.  If  the 
reactions  are  indefinite  they  should  be  repeated  with  the  immune  sera 
diluted  1  : 2  with  normal  salt  solution. 

Blake  has  found  a  precipitable  substance  in  the  peritoneal  exudate 
of  mice  used  in  the  differentiation  of  pneumococci  by  the  agglutination 
method  (see  page  308).  After  securing  the  exudate  it  is  thoroughly 
centrifuged,  and  equal  parts  (0.5  c.c.)  of  the  clear  supernatant  fluid  and 
antipneumococcus  sera  belonging  to  types  I  and  II  mixed  in  small  clean 
test-tubes.  The  development  of  a  ring  or  distinct  cloud  indicates  the 
type  of  infection  if  belonging  to  types  I  or  II,  and  the  method  saves 
time  as  compared  with  the  agglutination  test. 

Steinfield,  working  in  my  laboratory,  has  been  able  to  confirm  the 
work  of  Dochez,  Avery  and  Blake,  and  the  practical  value  of  these  tests. 

FLOCCULE-FORMING  REACTIONS 

Fornet  Ring  Test. — Owing  to  the  wonderful  activity  that  has  marked 
the  research  work  of  syphilis  several  precipitation  tests  for  diagnostic 
purposes  were  devised.  These  have  all  been  overshadowed  and  forsaken 
for  the  Wassermann  complement-fixation  test.  Fornet  applied  his  ring 
test,  using  the  serum  of  patients  with  manifest  luetic  symptoms  as  the 
precipitinogen,  and  the  serum  of  paretics  as  the  precipitating  or  immune 
serum.  Klausner  advocated  a  simple  test  consisting  of  mixing  in  a  small 
test-tube  0.2  c.c.  of  fresh,  active,  and  absolutely  clear  serum,  with  0.6 
c.c.  of  distilled  water.  This  serum  and  the  control  mixtures  are  allowed 
to  stand  at  room  temperature  for  from  seven  to  fifteen  hours,  when  a 
thick,  flocculent  precipitate  of  fibrin  globulin  will  appear  at  the  bottom 
of  the  tube. 

Porges-Meier  Reaction. — Forges  and  Meier  observed  that  luetic 
21 


322  PRECIPITINS 

serums  are  capable  of  producing  flocculent  precipitates  from  solutions 
of  lecithin  and  similar  salts.  Two-tenths  of  a  cubic  centimeter  of  a  1 
per  cent,  solution  of  Merck's  sodium  glycocholate  in  distilled  water  is 
placed  in  narrow  test-tubes,  and  an  equal  amount  of  the  patient's 
serum,  which  must  be  absolutely  clear  and  inactivated  by  heating  at 
56°  C.  for  thirty  minutes,  is  added.  This  mixture  and  the  known  nor- 
mal and  luetic  controls  are  kept  at  room  temperature  for  from  eighteen 
to  twenty-four  hours.  A  positive  reaction  is  marked  by  the  appearance 
of  distinct  coarse  flocculi,  mere  turbidity  or  faint  precipitation  being 
regarded  as  negative. 

Herman-Perutz  Reaction. — More  recently  Herman  and  Perutz  have 
devised  a  similar  test  requiring  the  following  two  solutions :  Solution  1 
(stock  solution,  diluted  1 :  20  with  distilled  water  before  use)  consists  of: 
Sodium  glycocholate,  2  gm.,  cholesterol,  0.4  gm.;  95  per  cent,  alcohol, 
100  c.c.  Solution  2  (freshly  prepared  before  use)  is  a  2  per  cent,  solu- 
tion of  sodium  glycocholate  in  distilled  water.  The  test  is  performed  by 
adding  to  0.4  c.c.  of  clear  inactive  serum  (heated  at  56°  C.  for  half  an 
hour)  in  a  small  test-tube  0.2  c.c.  of  solution  1  and  0.2  c.c.  of  solution  2. 
The  tubes  are  tightly  plugged  with  cotton  and  set  aside  at  room  tem- 
perature for  twenty-four  hours,  after  which  the  presence  or  absence  of 
precipitation  is  noted.  It  is  well  in  this  test,  as  in  all  immunologic  re- 
actions, to  prepare  controls  with  known  normal  and  luetic  serums  and 
with  distilled  water. 

None  of  these  reactions  has  been  found  specific,  and  none  has  been 
generally  adopted,  the  far  greater  accuracy  of  the  Wassermann  reaction 
having  made  this  method  more  valuable. 

Noguchi  Butyric-acid  Test. — Noguchi  has  devised  a  very  useful 
test  for  the  detection  of  an  increased  amount  of  protein,  particularly 
globulin,  in  cerebrospinal  and  other  body-fluids.  In  my  experience  this 
test  has  proved  of  particular  value  in  establishing  the  differential  diag- 
nosis between  serous  and  tuberculous  meningitis,  being  negative  in  the 
former  and  positive  in  the  latter,  whereas  in  both  the  fluid  may  be  clear, 
the  cytology  may  be  indefinite,  and  tubercle  bacilli  may  escape  detection. 
Serous  meningitis  is  not  a  true  infection,  but  a  reflex  vasomotor  disturb- 
ance of  the  cerebral  vessels,  causing  an  outpouring  of  serum  that  leads 
to  various  pressure  symptoms  closely  resembling  those  of  a  true  meningi- 
tis. This  condition  is  particularly  common  during  childhood,  and  the 
general  symptoms,  the  increased  pressure  of  the  cerebrospinal  fluid,  and 
its  clear,  watery  character,  are  features  that  resemble  those  of  tubercu- 
lous meningitis.  It  is  just  in  such  cases — and  they  are  frequent — that 
I  have  found  this  protein  reaction  of  considerable  value.  A  positive 


PRACTICAL    APPLICATIONS  323 

reaction  practically  always  means  a  true  meningitis;  a  negative  reaction 
usually  means  "  serous  meningitis,"  with  a  much  better  prognosis  if  the 
underlying  cause  is  corrected. 

Noguchi  has  found  the  test  positive  in  about  90  per  cent,  of  cases  of 
general  paralysis  and  in  60  per  cent,  of  cases  of  locomotor  ataxia  or  cere- 
bral or  spinal  syphilis.  In  the  diagnosis  of  syphilis  the  Wassermann 
reaction  with  cerebrospinal  fluid  has  greater  value  than  the  protein  re- 
action. However,  the  best  results  in  diagnosis  are  usually  secured  by  a 
Wassermann  test,  butyric-acid  test,  and  total  and  differential  cell-counts. 
In  a  case  where  the  diagnosis  rests  between  tuberculous  meningitis  and 
syphilis,  a  positive  butyric-acid  test  and  a  negative  Wassermann  reaction 
would  decide  in  favor  of  the  former. 

The  test  is  extremely  simple.  Into  a  small,  thin-walled  test-tube 
place  0.2  c.c.  of  cerebrospinal  fluid  (which  must  be  clear  and  free  from 
blood) ;  add  1  c.c.  of  a  10  per  cent,  solution  of  butyric  acid  in  normal  salt 
solution;  heat  over  a  low  flame  and  boil  for  a  short  period.  Then  add 
quickly  0.2  c.c.  of  a  normal  solution  of  sodium  hydroxid  and  boil  once 
more  for  a  few  seconds.  The  presence  of  an  increased  content  of  protein 
is  indicated  by  the  appearance  of  a  granular  or  flocculent  precipitate, 
which  gradually  settles  to  the  bottom  qf  the  tube,  under  a  clear  super- 
natant fluid  (Fig.  88). 

The  velocity  and  intensity  of  the  reaction  vary  with  the  quantity  of 
the  protein  contained  in  a  given  specimen.  The  granular  precipitate 
appears  within  a  few  minutes  in  a  specimen  containing  a  considerable 
increase  in  protein,  whereas  one  hour  may  be  required  to  obtain  a  dis- 
tinct reaction  in  specimens  weaker  in  protein.  In  obtaining  the  reaction, 
the  time  period  should  not  be  greater  than  two  hours.  A  faint  opales- 
cence  without  the  frrwotion  of  a  distinct  precipitate  is  to  be  regarded  as 
within  the  limits  of  the  normal. 

McDonagh  "Gel"  Test. — McDonagh1  has  recently  advocated  what 
he  has  designated  a  "gel"  test  for  the  diagnosis  of  syphilis.  Clear  and 
blood-free  sera  are  employed,  and  it  is  necessary  to  include  a  known  posi- 
tive and  negative  serum  each  time  the  tests  are  conducted.  Into  each 
of  three  dry  tubes  place  2,  3,  and  4  drops  of  serum,  respectively;  add  0.1 
c.c.  of  acetic  anhydrid  and  1  c.c.  of  glacial  acetic  acid;  the  tubes  are  now 
well  shaken  and  a  drop  of  a  saturated  watery  solution  of  ammonium 
sulphate  added.  Instead  of  acetic  anhydrid  and  solution  of  ammonium 
sulphate  the  test  may  be  conducted  with  0.2  c.c.  of  a  saturated  solution 
of  lanthanum  sulphate,  thorium  sulphate,  or  thorium  nitrate  in  glacial 
acetic  acid.  A  preliminary  reading  may  be  made  and  a  final  reading 
1  Brit.  Jour,  of  Dermat.,  1916,  April- June,  114. 


324 


PRECIPITINS 


after  the  tubes  have  stood  over  night.  "In  all  the  tubes  at  first  crystals 
form,  but  in  the  tubes  containing  normal  serum  they  often  disappear 
in  six  to  twenty-four  hours,  while  they  remain  in  the  tubes  with  syphilitic 
serum."  Strickler  has  used  this  test  in  my  laboratory,  and  my  obser- 
vations of  his  results  leads  me  to  the  conclusion  that  it  is  frequently 
difficult  to  interpret  the  reactions,  and  that  the  test  has  not  by  any  means 
the  practical  value  of  the  Wassermann  reaction. 


[  I    W 


FIG.  88. — THE  NOGTTCHI  BUTYRIC-ACID  TEST  FOR  GLOBULINS. 
The  tube  on  the  extreme  left  shows  the  formation  of  flocculi  within  a  few  minutes 
after  adding  NaOH;  the  middle  tube  shows  a  strongly  positive  reaction  after  stand- 
ing several  hours  (supernatant  fluid  quite  clear) ;  the  tube  on  the  extreme  right  shows 
a  very  slight  opalescence,  but  no  flocculi  (within  the  limits  of  normal). 

PROTEIN  PREOPITINS 

The  protein  precipitins  have  a  larger  range  of  value  and  represent 
one  of  the  most  important  practical  aids  in  forensic  medicine.  As  men- 
tioned elsewhere,  a  precipitating  immune  serum  reacts  only,  or  at  least 
best,  with  its  homologous  serum.  The  precipitin  reaction  is,  therefore, 
highly  specific,  and  offers  a  method  whereby  proteins  can  be  easily  and 
definitely  determined — a  problem  that  could  not  be  solved  by  chemistry. 

By  means  of  lactoserums  various  milks  and  cheeses  may  be  recognized 
and  their  source  determined. 

By  using  appropriate  immune  serums  seminal  fluids  and  stains  may 
be  detected,  and  in  medicolegal  cases,  where  the  question  is  one  of  rape, 
this  reaction  possesses  considerable  value  in  differentiating  seminal  from 
leukorrheal  stains. 


PRACTICAL   APPLICATIONS 


325 


The  precipitin  reaction  is  likewise  of  great  value  in  rnedicolegal  cases 
in  determining  the  source  of  blood-stains,  the  original  application  and 
technic  having  been  largely  worked  out  by  Wassermann,  Schutze,  Uh- 
lenhuth,  and  Weidanz.  Thus  in  a  case  where,  for  example,  a  bloody 
towel  is  found  in  the  possession  of  a  man  charged  with  murder,  the  prose- 
cution may  see  in  this  a  proof  of  crime,  whereas  the  defendant  may  claim 
that  the  stains  are  those  of  dog's  blood.  Microscopic  and  chemical 
tests  may  show  that  the  stains  are  blood-stains,  but  they  cannot  deter- 
mine their  source.  The  blood-stained  towel  is  placed  in  water  or  salt 
solution,  and  a  portion  of  the  extract  is  mixed  with  the  serum  of  a  rabbit- 
immunized  against  human  serum,  and  another  portion  with  the  serum  of 
a  rabbit  immunized  against  dog's  serum.  If  the  first  mixture  shows  a 
precipitate,  the  stain  was  made  by  blood  from  a  human  being;  if,  on 
the  other  hand,  this  mixture  remains  clear  and  the  second  shows  a  pre- 
cipitate, this  is  strongly  indicative  of  the  presence  of  dog's  blood. 

This  method  has  also  cleared  up  a  number  of  scientific  problems, 
especially  that  of  showing  the  blood  relationship  of  man  and  the  lower 
animals.  Just  as  a  group  agglutination  demonstrates  the  close  relation- 
ship existing  between  various  bacteria,  so,  also,  serum  precipitins  prove 
that  a  distinct  relationship  exists  between  the  different  species  of  animals. 

For  example,  an  antihuman  serum  in  low  dilution  will  precipitate  the 
serum  of  monkeys.  The  differentiation  between  human  and  monkey 
serum  can  be  accomplished,  however,  by  immunizing  the  monkey  with 
human  serum,  when  a  precipitin  is  formed  that'  reacts  with  human  serum 
alone,  an  isoprecipitin,  or  one  active  against  the  monkey's  own  serum, 
not  being  developed  as  a  general  rule. 

The  precipitin  tests  are  likewise  of  value  in  food  inspection,  as,  for 
instance,  to  determine  the  nature  of  meats.  For  example,  in  order  to 
detect  the  presence  of  dog  or  horse  flesh  in  sausage,  extracts  of  the  sausage 
are  made  and  tested  with  anti-dog  and  anti-horse  serum,  the  presence  of 
precipitates  indicating  strongly  the  presence  of  the  meat  of  these  animals. 
With  an  appropriate  technic  even  salted  and  cooked  flesh  may  be  recog- 
nized, although  when  the  meat  has  been  cooked  it  is  necessary  to  prepare 
immune  serums  by  immunizing  rabbits  with  extracts  of  cooked  meats. 

In  this  connection  it  may  be  stated  that  specific  organic  reactions 
have  been  secured  by  various  investigators  by  prolonged  immunization 
of  rabbits  with  certain  organ  extracts.  Thus  it  is  possible  to  differen- 
tiate between  the  liver  and  kidney  of  the  same  animals;  such  tests  have, 
however,  but  limited  practical  value.  Maragliano  attempted  to  apply 
this  test  of  organic  specificity  to  the  serodiagnosis  of  malignant  tumors 
by  preparing  immune  serums  by  the  injection  of  tumor  juices,  securing  a 


326  PRECIPITINS 

serum  that  yielded  a  precipitate  with  the  albumins  of  a  cancerous  tumor. 
The  test  is  not  absolutely  specific,  and  its  practical  value  in  diagnosis 
requires  confirmation. 

Freund  and  Kaminer  have  described  a  precipitin  reaction  in  cancer 
with  an  extract  of  cancer  tissue,  but  the  practical  value  of  the  test  has 
not  been  established. 


TECHNIC  OF  PREdPITIN  REACTIONS 
DIFFERENTIATION  OF  HUMAN  AND  ANIMAL  BLOOD — BIOLOGIC  BLOOD  TEST 

Unless  a  stain  is  definitely  known  to  be  a  blood-stain  it  is  necessary 
to  establish  its  identity  by  making  a  chemical  test  before  proceeding  with 
the  precipitin  reactions.  For  example,  old  stains  upon  clothing  may  be 
due  to  substances  other  than  blood,  such  as  coffee  and  fruit-juices. 
Blood-stains  upon  clothing,  metal,  wood,  or  glass  may  be  used  for  making 
these  reactions  and  their  source  determined. 

To  identify  the  stain  as  one  of  blood,  a  portion  may  be  taken  into 
solution  in  distilled  water,  rendered  slightly  acid  with  dilute  acetic  acid, 
filtered  until  clear,  and  examined  spectroscopically.  Or  the  Teichmann 
hemin  crystal  test  may  be  applied  to  the  stain  by  transferring  to  a  clean 
slide  a  small  amount  of  material  scraped  from  the  stain;  add  a  few  small 
crystals  of  sodium  chlorid,  crush  the  crystals,  and  mix  the  powder  with 
the  dry  material.  Place  a  clean  cover-glass  over  the  stained  material 
and  run  a  small  amount  of  glacial  acetic  acid  under  the  cover-glass. 
Heat  the  preparation  to  just  about  the  boiling-point  for  a  minute,  re- 
plenishing the  acid  as  may  be  necessary.  The  fluid  turns  brown. 
The  specimen  is  allowed  to  cool  a  few  minutes,  and  is  then  examined 
microscopically  for  the  presence  of  brown  rhombic  crystals  of  hemin 
(Fig.  89).  It  may  be  necessary  to  reheat  the  specimen  several  times 
before  the  crystals  are  obtained.  With  stains  in  cloth  and  particularly 
those  partially  removed  by  washing,  other  chemical  methods  for  detec- 
tion, as  the  guaiac,  benzidin,  or  Furth1  leukomalachite-green  tests,  must 
be  employed. 

Having  shown  that  a  given  stain  is  actually  a  blood-stain,  the  source 
of  the  blood  may  be  determined  as  the  result  of  the  precipitin  reaction, 
which  consists  in  extracting  the  stain  in  normal  salt  solution  and  mixing 
with  antiserums  prepared  by  immunizing  rabbits  with  human  and  various 
animal  serums.  Since  the  antiserums  are  known,  a  precipitate  with  any  ' 
one  of  the  extracts  indicates  that  the  blood  in  the  stain  was  derived  from 

1  Fiirth-Smith,  Physiological  and  Pathological  Chemistry  of  Metabolism,  1916, 
Lippincott  &  Co.,  546. 


FIG.  89. — HEMIN  CRYSTALS. 

Prepared  after  the  method  described  in  the  text.     From  a  stain  (over  two  months 
old)  of  sheep  blood  on  a  towel. 


FIG.  90. — PRECIPITIN  REACTION.     PREPARATION  OF  EXTRACT  OF  BLOOD-STAIN. 

The  tube  on  the  left  shows  the  color  of  an  extract  of  a  blood-stain;  the  middle 
tube  shows  this  extract  so  diluted  as  to  yield  a  faint  albumin  reaction  with  nitric  acid; 
the  tube  on  the  right  shows  the  foam  test  with  the  same  diluted  extract  (about  1 :1000). 


TECHNIC    OF    PRECIPITIN    REACTIONS  327 

the  same  species  of  animal.  The  reaction  is  based  upon  the  principle  of 
the  specificity  of  antigen  and  its  antibody.  Here  the  antibody  is  known, 
and  is  used  in  the  test  to  detect  the  antigen. 

As  mentioned  elsewhere,  because  of  the  presence  of  group  precipitins 
the  reaction  is  fraught  with  certain  technical  difficulties  of  importance, 
especially  in  medicolegal  cases.  In  most  instances  it  may  suffice  to  show 
that  a  stain  is  of  human  blood,  as  will  be  indicated  by  a  strong  reaction 
with  human  blood  and  negative  reactions  with  the  bloods  of  lower  ani- 
mals. If  the  reaction  is  negative  with  antihuman  serum,  the  antiserums 
of  the  domestic  animals,  such  as  that  of  the  dog,  cat,  hog,  ox,  horse,  etc., 
are  tried.  Although  the  blood  of  the  higher  apes,  and  even  of  the  lower 
orders  of  monkeys,  may  react  slightly  with  human  blood,  this  factor  may 
be  determined  by  observing  a  proper  technic  of  dilution,  or  the  possibil- 
ity of  a  given  stain  being  one  of  monkey  blood  being  definitely  worked  out. 

The  reaction  can  be  obtained  from  blood  in  an  advanced  state  of 
putrefaction,  or  from  a  stain  that  has  been  dried  for  a  year  or  more. 
Tests  with  mummies,  however,  have  reacted  negatively,  and  stains  or 
clots  altered  by  heat  may  not  react  unless  the  antiserum  has  been  pre- 
pared with  a  similar  antigen. 

This  same  technic  may  be  employed  for  the  recognition  of  seminal 
stains,  especially  in  cases  of  rape.  The  stain  is  taken  into  solution  in 
exactly  the  same  manner  as  the  blood-stain,  and  tested  with  an  anti- 
human  semen  serum  prepared  by  immunizing  rabbits  with  human  semen. 

Preparation  of  the  Extract  of  Stain  (the  Precipitinogen).— If  the 
stain  is  on  clothing,  a  portion  three  inches  square  should  be  carefully 
torn  into  shreds  with  forceps  and  scissors,  and  not  with  the  fingers, 
and  placed  in  40  c.c.  of  normal  salt  solution.  If  the  stain  is  upon  wood, 
glass,  or  metal,  the  staining  substance  should  be  carefully  scraped  off 
with  a  knife  and  placed  in  the  salt  solution.  As  a  further  control  on  the 
technic  an  unstained  portion  of  the  clothing  should  be  extracted  in  the 
same  manner,  in  order  to  show  that  the  latter  alone  does  not  give  the 
reaction.  The  mixtures  must  not  be  shaken,  but  should  be  stood  aside 
for  from  two  to  twenty-four  hours,  depending  upon  the  rapidity  of  ex- 
traction. The  extract  should  preferably  not  be  stronger  than  1 :  1000. 
The  strength  may  be  estimated  by  removing  1  c.c.  of  the  extract  into 
another  tube,  diluting  with  from  10  to  20  c.c.  of  normal  salt  solution,  and 
gently  shaking.  If  a  persistent  froth  appears  upon  the  surface  of  the 
fluid,  sufficient  extraction  has  occurred.  Place  2  c.c.  in  a  test-tube,  heat 
to  boiling,  and  add  a  drop  of  a  25  per  cent,  solution  of  nitric  acid.  A 
faint  opalescence  indicates  that  the  strength  of  the  extract  is  about  1 : 1000 
(Fig.  90).  If  a  heavy  precipitate  forms,  the  amount  of  normal  salt  solu- 


328 


PRECIPITINS 


tion  that  must  be  added  to  a  portion  of  the  extract  to  reduce  it  to  a 
dilution  of  1 :  1000  should  be  determined.  The  extract  should  be  almost 
colorless  by  transmitted  light,  and  must  be  crystal  clear;  this  may  be  ac- 
complished by  filtering  it  through  a  clean  sterile  Berkefeld  filter.  It  is 
highly  desirable  that  the  extract  be  sterile,  as  the  reaction  may  require 


FIG.  91. — AN  UHLENHUTH  FILTER. 

Serum  is  poured  into  the  glass  cylinder  surrounding  the  "candle";  a  vacuum  is 
produced  by  the  water-pump;  the  nitrate  is  collected  in  the  small  bottle  attached  by 
rubber  tubing  to  the  graduated  cylinder  at  the  lower  portion  of  the  apparatus.  This 
filter  is  especially  adapted  for  filtering  small  amounts  of  serum  or  other  fluid. 

several  hours,  and  cloudiness  due  to  bacterial  growth  may  interfere  with 
the  result. 

The  Immune  Serum. — A  highly  potent,  sterile,  and  absolutely  crys- 
tal-clear serum  immune  against  the  protein  to  be  recognized  must  be 
prepared.  The  method  of  preparation  is  given  in  the  chapter  on  Active 
Immunization  of  Animals.  For  the  recognition  of  blood-stains  it  is  not 


TECHNIC    OF    PRECIPITIN    REACTIONS  329 

necessary  that  whole  blood  be  injected,  as  the  serum  alone  will  suffice. 
It  is  better  to  inject  a  number  of  rabbits  with  each  serum  and  to  give  all 
injections  intravenously.  From  the  third  injection  on,  preliminary 
titrations  are  made  according  to  the  technic  to  be  described  later,  as 
many  animals  succumb  after  a  large  number  of  injections  have  been 
given  them. 

The  serum  must  be  absolutely  clear.  Animals  should  be  bled  after 
a  period  of  fasting,  as  the  opalescence  of  the  serum  following  feeding 
cannot  be  removed  by  filtration  and  will  interfere  with  the  reaction. 
Precipitin  immune  serum  should  be  collected  with  a  scrupulous  aseptic 
technic,  and  stored  in  ampules  holding  1  c.c.  Although  it  is  best  not  to 
add  a  preservative,  the  addition  of  0.1  c.c.  of  a  1  per  cent,  solution  of 


FIG.  92. — TEST-TUBE  RACK  FOR  PRECIPITIN  AND  AGGLUTINATION  REACTIONS. 
The  strips  of  black  material  in  the  rear  of  the  tubes  facilitate  reading  the  reactions. 

phenol  to  each  cubic  centimeter  of  serum  does  not  render  the  fluid  cloudy 
and  aids  greatly  in  its  preservation. 

If,  after  long  standing,  a  precipitate  has  become  deposited  in  an  anti- 
serum,  this  should  not  be  shaken  up,  but  the  ampule  should  be  carefully 
opened  and  the  clear  supernatant  serum  drawn  off  with  a  capillary  pipet. 
A  serum  that  has  become  cloudy  may  be  cleared  partially  or  entirely  by 
filtering  it  through  a  small  candle  filter,  although  even  an  infected  and 
offensive  serum  will  give  the  reaction  (Fig.  91). 

Apparatus. — Long  and  narrow  test-tubes,  10  cm.  by  0.8  cm.,  are 
used.  These  must  be  absolutely  clean,  and  preferably  sterile. 

The  test-tube  rack  devised  by  Uhlenhuth,  in  which  the  tubes  hang 
suspended  in  beveled  holes,  is  quite  satisfactory.  Where  a  test  is  carried 
out  with  many  controls,  a  rack  similar  to  the  one  shown  in  the  illustra- 
tion (Fig.  92)  is  quite  serviceable.  A  strip  of  black  material  placed  be- 


330 


PRECIPITINS 


hind  the  tubes  aids  greatly  in  the  detection  of  the  finer  degrees  of  opal- 
escence  or  precipitation. 

Preliminary  Titrations. — The  precipitin  content  of  an  immune  serum 
is  titrated  frequently  during  the  process  of  immunization  and  after  the 
animal  has  been  bled.  For  medicolegal  purposes,  Uhlenhuth  advises 
the  use  of  only  highly  valent  serums.  He  considers  an  antiserum  ef- 


. 


FlG.   93. — TlTRATION  OF  A  PRECIPITIN  (SERUM). 

Not  all  tubes  of  the  series  are  here  shown.  Note  the  well-marked  precipitate  in 
the  bottom  of  the  first  two  tubes  (1 :  100  and  1 :  500);  the  third  tube  (1 :  1000) 
shows  less  precipitate;  the  fourth  tube  (1  :  2000)  is  negative  (clear  and  no  precipi- 
tate). The  titer  of  this  serum  was  recorded  as  1 :  1000. 


ficient  if  0.1  c.c.  of  it,  when  added  to  its  respective  serum-antigen 
diluted  1 : 1000,  produces  a  distinct  turbidity,  either  at  once  or  in  from 
one  to  five  minutes  at  the  latest. 

Into  a  series  of  six  test-tubes  place  2.0  c.c.  of  the  following  dilutions 
of  serum-antigen,  prepared  with  normal  salt  solution:  1:100,  1:500, 
1:1000,  1:2000,  1:5000,  and  1:10,000.  To  each  tube  add  0.1  c.c.  of 


TECHNIC    OF    PRECIPITIN    REACTIONS  331 

clear  immune  serum.  The  tubes  must  not  be  shaken.  Within  from  one 
to  five  minutes  a  faint,  misty  cloud  appears  at  the  bottom  of  the  tubes 
reacting  positively,  and  this  becomes  a  distinct  precipitate  within  one- 
half  to  one  hour  (Fig.  93). 

Before  performing  the  actual  test  with  the  unknown  blood-stain  it 
is  advisable  to  test  the  entire  reaction  with  a  similar  known  blood-stain 
in  order  to  make  sure  that  all  ingredients  are  in  good  working  order. 
In  laboratories  equipped  for  medicolegal  examinations  stains  upon  linen, 
as  by  the  blood  of  man,  dog,  cat,  ox,  horse,  etc.,  and  their  respective 
antiserums  are  always  kept  in  readiness  for  making  the  preliminary  and 
actual  tests. 

Technic  of  the  Test. — The  following  mixtures  are  set  up  in  a  series  of 

test-tubes.     Fresh  sterile  pipets  should  be  used  in  handling  the  various 

solutions.     The  immune  serum  should  be  added  slowly,  and  in  such  a 

way  that  it  will  flow  down  the  side  of  the  tube  and  collect  at  the  bottom. 

Tube  1:    2  c.c.  of  extract  of  unknown  substance  in  dilution  of 

1:1000+0.1  c.c.  of  immune  serum. 
Tube  2:   2  c.c.  of  the  unknown  extract  in  dilution  of  1  :  5000+0.1 

c.c.  of  immune  serum. 
Tube  3:  2  c.c.  of  the  unknown  extract  in  dilution  of  1  :  10,000+0.1 

c.c.  of  immune  serum. 
Tube  4:  2  c.c.  of  the  unknown  extract  in  dilution  ofl:100+0.1c.c. 

of  normal  rabbit  serum  (control). 

Tube  5:  2  c.c.  of  a  1:1000  dilution  of  the  serum  of  that  species  of 
animal  whose  blood  is  suspected  to  be  present  in  the  unknown 
solution +0.1  c.c.  of  immune  serum  (control). 

Tube  6:  2  c.c.  of  the  extract  of  unknown  substance  alone  (control). 
Tube  7:   0.1  c.c.  of  the  immune  serum+2  c.c.  of  normal  salt  solu- 
tion (control). 
Tube  8:  2  c.c.  of  the  extract  of  the  unstained  portion  of  clothing + 

0.1  c.c.  of  immune  serum  (control). 

The  tubes  are  not  shaken,  are  kept  at  room  temperature,  and  the 
results  are  read  after  from  ten  to  twenty  minutes.  Exposure  to  incu- 
bator temperature  facilitates  the  reaction.  With  proper  immune  serums, 
and  especially  in  medicolegal  work,  a  positive  reaction  should  appear 
within  two  to  five  minutes  as  a  faint,  misty  cloud  at  the  bottom  of  the 
test-tube.  Within  five  minutes  this  becomes  more  definite,  and  in  from 
ten  to  twenty  minutes  the  precipitate  is  seen  (Fig.  94).  Any  cloudi- 
ness that  develops  later  than  twenty  minutes  after  the  beginning  of  the 
reaction  has  no  significance. 


332 


PRECIPITINS 


In  tests  other  than  those  employed  in  medicolegal  work,  especially 
if  the  antiserums  are  weaker  than  desired,  the  reaction  may  be  read  after 
one  to  two  hours. 

In  the  foregoing  test,  if  positive  results  are  obtained  in  tubes  1,  2,  or 


FIG.  94. — A  PRECIPITIN  REACTION — BIOLOGIC  BLOOD  TEST. 

This  reaction  was  set  up  with  an  extract  of  a  stain  of  sheep  blood  on  a  towel, 
over  three  months  of  age,  with  an  anti-sheep  immune  serum. 

The  first  tube,  containing  a  1  :  1000  dilution  of  the  blood  (extreme  left),  shows  a 
well-marked  precipitate;  in  the  second  (1  :  2000)  the  reaction  is  also  well  marked; 
the  third  (1  :  5000)  shows  no  precipitate;  the  fourth  contains  a  1  :  1000  dilution  of 
blood-stain,  with  normal  rabbit  serum  as  a  control,  and  shows  no  precipitate;  the 
fifth  is  the  positive  control  with  known  sheep  blood  extract  and  immune  serum;  the 
last  tube  is  No.  8  of  the  series,  and  contains  an  extract  of  the  non-bloody  portion  of 
the  towel  with  immune  serum;  no  precipitate. 

3  and  tube  5,  and  all  the  others  react  negatively,  the  presence  of  the  blood 
or  protein  of  the  species  suspected  in  the  unknown  extract  is  established. 
If  the  entire  test  proves  negative,  the  species  to  which  the  unknown 
specimen  belongs  must  be  determined  with  new  antiserums  prepared  for 
each  species,  and  the  tests  conducted  in  the  manner  described. 


TECHNIC    OF    PRECIPITIN    REACTIONS  333 

Partial  reactions  between  closely  related  species  due  to  group  pre- 
cipitins  seldom  occur,  and  are  easily  detected  when  the  technic  described 
is  employed.  The  precipitin  test,  as  determined  by  the  extensive  ex- 
perience of  Nuttall,  is  highly  specific,  and  it  is  only  between  very  closely 
related  animals,  such  as  the  hare  and  the  rabbit,  the  horse  and  the 
mule,  the  sheep  and  the  goat,  etc.,  that  any  doubt  can  arise. 

When  only  very  limited  amounts  of  the  unknown  stain  are  available, 
the  test,  according  to  Hauser,  can  be  carried  out  in  slender,  clean,  and 
sterile  capillary  tubes.  The  piece  of  stained  clothing  is  torn  into  shreds, 
extracted,  and  filtered  until  clear.  The  tests  are  performed  by  drawing 
a  small  amount  of  the  unknown  solution  into  a  capillary  tube,  and  under- 
lying this  with  a  small  amount  of  immune  serum.  As  many  controls 
as  possible  are  put  up  in  the  same  manner.  A  distinct  whitish  ring  will 
form  in  the  positive  tubes  at  the  line  of  contact  between  the  two  fluids; 
this  is  best  seen  by  holding  the  tube  against  a  black  background. 

DETECTION  OF  MEAT  ADULTERATION 

The  principle  of  this  method  is  the  same  as  that  in  the  foregoing  test. 
An  extract  of  a  meat  will  yield  a  precipitate  when  mixed  with  its  anti- 
serum,  prepared  by  immunizing  rabbits  with  an  extract  of  the  flesh  or 
with  the  blood-serum  of  some  other  animal. 

The  method  is  especially  serviceable  in  food  inspection  for  the  de- 
tection of  horse,  dog,  or  other  foreign  flesh  in  meat  mixtures,  such  as 
sausage  and  the  like.  Even  salted  and  cooked  meats  may  be  used  in  the 
test,  although  the  latter  may  require  the  use  of  antiserums  prepared  by 
immunizing  with  heated  or  cooked  antigen. 

Preparation  of  the  Meat  Extract. — To  prepare  this,  about  50  grams 
of  flesh  are  removed  from  the  deeper  parts  of  the  specimen  by  means  of  a 
sterile  knife,  and  through  a  fresh  opening,  as  this  portion  has  been  least 
exposed  to  the  methods  of  preservation,  especially  at  the  high  tempera- 
tures to  which  the  meat  may  have  been  exposed.  It  should  contain  as 
little  fat  as  possible.  It  is  then  placed  on  a  clean  sterile  tile  and  cut  into 
smaller  pieces,  and  finally  minced  by  passing  it  through  a  perfectly  dean 
meat-grinder  or  chopping  it  with  a  sterile  chopping  knife.  After  being 
finely  minced  the  meat  is  placed  in  a  sterile  Erlenmeyer  flask,  and  cov- 
ered with  100  c.c.  of  sterile  normal  salt  solution.  The  mixture  of  meat 
and  salt  solution  is  kept  for  about  six  hours  at  room  temperature,  or 
overnight  in  the  refrigerator,  the  flask  being  gently  shaken  from  time  to 
time. 

Salted  meat  should  be  ground  and  freshened  by  placing  it  in  a  large 


334  PRECIPITINS 

sterile  Erlenmeyer  flask  and  covering  it  with  sterile  distilled  water,  re- 
newed several  times  in  the  course  of  fifteen  minutes  without  shaking  the 
flask. 

Since  the  presence  of  a  great  deal  of  fat  interferes  with  the  reaction, 
it  is  advisable  to  remove  it  beforehand  by  extracting  it  with  ether  and 
chloroform  for  from  twelve  to  twenty-four  hours  (Miessner  and  Herbst). 
Pork  sausages  are  usually  quite  fatty,  and  may  require  this  preliminary 
treatment.  To  make  the  extraction,  take  about  75  to  100  grams  of  the 
minced  meat  or  sausage  and  place  it  in  a  large  Erlenmeyer  flask  and  cover 
with  equal  parts  of  ether  and  chloroform.  After  twenty  hours  the  ether 
and  chloroform  are  poured  off,  the  meat  is  washed  once  or  twice  with 
sterile  normal  salt  solution,  and  then  extracted  hi  100  c.c.  of  salt  solu- 
tion, as  described  elsewhere. 

To  determine  whether  a  sufficient  quantity  of  protein  substances  has 
passed  into  solution  place  2  c.c.  in  a  test-tube  and  shake  vigorously.  If 
a  foam  develops  and  persists  for  some  time,  the  extraction  may  be  said 
to  be  complete.  It  must  then  be  filtered  until  it  becomes  perfectly  clear. 
With  extracts  of  fresh  lean  meat  this  is  usually  accomplished  by  filtering 
through  a  hard  filter-paper  moistened  with  salt  solution.  If  it  is  not 
crystal  clear,  and  especially  if  the  meat  to  be  examined  is  fat  or  salt,  it 
may  be  necessary  to  filter  through  a  sterile  Berkef eld  filter. 

To  make  the  test  the  extract  should  contain  about  one  part  of  pro- 
tein in  500  parts  of  salt  solution.  To  determine  this,  2  c.c.  of  the  clear 
filtrate  are  placed  in  a  test-tube  and  heated,  a  drop  of  dilute  nitric  acid 
being  added.  If  a  marked  cloudiness  and  a  flocculent  precipitate  de- 
velops, the  extract  is  too  concentrated  and  must  be  diluted  with  clear 
normal  salt  solution  until  the  heat  and  acid  test  causes  only  a  diffuse, 
opalescent  cloudiness  that  settles  at  the  bottom  of  the  tube  after  five 
minutes  as  a  slight  precipitate. 

Before  proceeding  with  the  experiment  the  reaction  of  the  solution 
should  be  tested  with  litmus  paper,  and  if  it  is  found  to  be  acid,  it  should 
be  neutralized  very  carefully  with  ^  sodium  hydroxid. 

Extracts  of  the  meats  that  are  known  or  likely  to  be  present,  such  as 
extracts  of  pork  and  beef,  should  be  prepared  as  controls. 

Preparation  of  Immune  Serum. — An  immune  serum  against  that 
variety  of  flesh  that  is  to  be  determined  in  the  unknown  specimen  is 
prepared  by  injecting  rabbits  intravenously  with  the  serum  of  an  ani- 
mal of  that  species.  For  example,  if  the  object  is  to  test  for  dog  meat, 
an  anti-dog  serum  is  prepared  by  immunizing  rabbits  with  sterile  dog 
serum. 


TECHNIC    OF   PRECIPITIN   REACTIONS  335 

As  has  repeatedly  been  mentioned,  it  is  advisable  to  immunize  a 
number  of  rabbits  at  the  same  time,  for  only  a  small  number  will  yield  a 
satisfactory  serum  after  the  third  injection. 

Immunization  may  be  performed  with  extracts  of  flesh  that  have  been 
filtered  and  heated  at  56°  C.  for  an  hour  to  secure  partial  sterilization. 
Such  injections,  when  given  subcutaneously,  are  likely  to  produce  ex- 
tensive sloughing,  and  with  any  method  of  immunization  the  mortality 
is  high. 

After  the  third  inoculation  it  is  well  to  remove  a  small  amount  of 
blood  from  the  ear  and  make  a  preliminary  titration.  This  is  performed 
in  exactly  the  same  manner  as  in  making  the  forensic  blood  test  pre- 
viously described.  An  antiserum  is  satisfactory  if  0.1  c.c.  produces  a 
well-marked  cloudiness  and  a  precipitate  in  ten  minutes  with  2  c.c.  of  a 
1 : 1000  dilution  of  the  serum  or  extract  of  flesh. 

In  addition  to  being  highly  potent,  the  immune  serum  must  be  crystal 
clear  and  sterile.  To  avoid  opalescence  the  animal  should  be  bled  only 
after  a  period  of  fasting. 

Technic. — If,  for  example,  the  object  is  to  determine  whether  a 
piece  of  meat  is  horse  flesh  or,  if  sausage,  contains  the  meat  of  this  ani- 
mal the  test  is  conducted  as  follows : 

Tube  1:   2  c.c.  of  unknown  extract,  1:500+0.1  c.c.  of  antihorse 

serum. 
Tube  2:   2  c.c.  of  unknown  extract,  1  : 1000+0.1  c.c  of  antihorse 

serum. 
Tube  3:   2  c.c.  of  unknown  extract,  1 : 5000 +0.1  c.c.  of  antihorse 

serum. 
Tube  4:  2  c.c.  of  horse  flesh  extract,  1 : 1000+0.1  c.c.  of  antihorse 

serum  (positive  control). 
Tube  5:    2  c.c.  of  unknown   extract,   1:500+0.1  c.c.  of  normal 

rabbit  serum. 

Tube  6:  2  c.c.  of  pork  extract,  1 : 500+0.1  c.c.  of  antihorse  serum. 
Tube  7:  2  c.c.  of  beef  extract,  1 : 500+0.1  c.c.  of  antihorse  serum. 
Tube  8:  2  c.c.  of  unknown  extract. 

Tube  9:   2  c.c.  of  sterile  salt  solution+0.1  c.c.  of  antihorse  serum. 
The  immune  serum  is  added  to  each  tube  very  carefully  and  run 
down  the  sides  of  the  tube,  rather  than  dropped  into  them.     The  tubes 
should  not  be  shaken. 

If  the  preliminary  titration  of  the  immune  serum  fulfils  the  ideal 
requirement  of  yielding  a  well-marked  cloudiness  within  five  to  ten 
minutes  with  a  1 : 1000  extract,  the  foregoing  test  should  be  recorded  at 


336  PRECIPITINS 

the  end  of  half  an  hour  at  room  temperature.  If  in  tubes  1,  2,  4,  and 
possibly  3  a  misty  cloudiness  should  appear  within  five  minutes,  the 
extract  is  very  probably  one  of  horse  flesh.  If  a  definite  precipitate 
forms  within  thirty  minutes,  the  other  tubes  remaining  clear,  horse 
flesh  or  the  flesh  of  some  other  single-toed  animal  is  present. 

If  the  preliminary  titration  does  not  show  a  precipitate  with  the 
immune  serum  until  at  the  end  of  one  or  two  hours,  this  interval  may  be 
utilized  for  conducting  the  test. 

In  a  similar  manner  tests  may  be  made  for  the  meat  of  dogs,  cats, 
or  any  other  animals  if  the  respective  immune  serums  are  used  with  the 
extract. 

BACTERIAL  PRECIPITINS 

As  has  previously  been  stated,  these  precipitins  have  slight  diagnostic 
significance,  as  the  information  they  yield  in  the  diagnosis  of  an  infec- 
tion or  in  the  differentiation  of  bacterial  species  may  be  gained  much 
more  easily  with  the  agglutinin  test. 

Bacterial  precipitinogens  are  prepared  by  filtering  ten  to  twenty-one 
day  bouillon  cultures  through  Berkefeld  filters.  The  filtrates  must  be 
absolutely  clear  and  sterile,  for  the  reaction  frequently  requires  a  num- 
ber of  hours,  and  if  bacteria  are  present,  they  may  grow  quickly,  produce 
turbidity,  and  mask  a  reaction. 

Immune  Serum. — This  is  prepared  according  to  the  technic  described 
under  Active  Immunization.  Rabbits  are  given  intravenous  injections 
of  increasing  doses  of  cultures  of  the  bacteria  themselves  or  of  filtrates, 
the  inoculum  being  heated  at  60°  C.  for  an  hour  previous  to  making  the 
injection.  After  the  third  dose  the  serum  is  titrated  and  the  injections 
continued  unless  the  serum  is  satisfactory. 

Technic. — A  known  quantity  of  precipitinogen  and  varying  amounts 
of  immune  serum  are  employed.  If  too  much  precipitinogen  is  fur- 
nished, the  precipitate  will  not  form,  and  one  that  has  already  formed 
may  dissolve  on  the  addition  of  more  precipitinogen. 

If,  for  example,  one  desires  to  determine  if  typhoid  precipitin  is 
present  in  a  given  serum,  the  test  is  conducted  as  follows: 

Tube  1:   2  c.c.  of  typhoid  bouillon  filtrate +0.05  c.c.  of  unknown 

serum +0.9  c.c.  of  normal  salt  solution 
Tube  2:    2  c.c.  of  typhoid  bouillon  filtrate +0.1  c.c.  of  unknown 

serum+0.9  c.c.  of  normal  salt  solution. 

Tube  3:    2  c.c.  of  typhoid  bouillon  filtrate+0.5  c.c.  of  unknown 
serum+0.5  c.c.  of  normal  salt  solution. 


TECHNIC    OF   PRECIPITIN   REACTIONS  337 

Tube  4:  2  c.c.  of  typhoid  bouillon  filtrate+1  c.c.  of  unknown  serum. 
Tube  5:   2  c.c.  of  typhoid  bouillon  filtrate-f-1  c.c.  of  typhoid  im- 
mune serum  (positive  control). 
Tube  6:   2  c.c.  of  typhoid  bouillon  filtrate+1  c.c.  of  normal  salt 

solution. 

Tube  7:  2  c.c.  of  typhoid  bouillon  filtrate+1  c.c.  of  normal  serum. 
Tube  8:  1  c.c.  of  serum+1  c.c.  of  normal  salt  solution. 
The  tubes  are  not  shaken,  and  are  kept  at  room  temperature  for 
from  one  to  six  hours.     If  the  unknown  serum  contains  considerable 
typhoid  precipitins,  a  positive  reaction  will  be  noticed  in  the  first  four 
tubes  in  a  short  time — often  within  from  ten  to  fifteen  minutes.     Tube 
5  should  show  a  strong  reaction  and  the  other  tubes  should  remain  clear. 
In  studying  the  biologic  relationship  of  an  organism  to  others  of  the 
same  group  its  immune  serum  may  be  used  in  amounts  of  1  c.c.  of  vary- 
ing  dilutions,    as   1:50,    1:100,    1:500,    1:1000,    1:2000,    1:4000,  and 
so  on,  with  a  constant  dose  of  1  or  2  c.c.  of  the  bouillon  filtrates  of  the 
various  organisms  studied.     A  comparison  of  the  precipitates  in  the 
respective  dilutions  of  the  different  filtrates  indicates  the  relationship, 
according  to  the  amount  of  group  precipitins  present  in  the  immune 
serum. 

22 


CHAPTER  XVIII 
CYTOLYSINS 

Amboccptors  and  Complements 

THE  cytolysins  include  a  number  of  antibodies  of  considerable  diag- 
nostic and  therapeutic  importance,  for  example,  the  hemolysins  and  the 
bacteriolysins.  It  will  be  remembered  that  the  various  antibodies  act 
differently  upon  their  antigens,  and  that,  according  to  the  side-chain 
theory,  as  their  antigens  become  more  highly  organized,  their  structure 
becomes  more  complicated.  For  example,  the  molecule  of  a  soluble 
toxin  may  be  considered  as  simple  in  structure,  and  accordingly  its 
antibody  has  been  conceived  as  being  likewise  simple,  and  composed  of  a 
plain  cast-off  receptor  or  side-arm  that  unites  directly  with  the  toxin 
and  neutralizes  it  without  further  aid.  Antitoxins  and  antiferments 
are  antibodies  of  this  nature.  For  more  highly  organized  antigens, 
however,  so  simple  an  antibody  will  not  suffice,  and  we  now  find  a  more 
complicated  antibody,  composed  of  a  portion  that  unites  with  the  anti- 
gen and  another  portion,  an  integral  part  of  the  antibody,  that  exerts  a 
special  selective  action  upon  the  antigen,  and  either  neutralizes  its  activ- 
ity or  prepares  it  for  ultimate  destruction.  To  this  class  of  antibodies 
belong  the  agglutinins  and  precipitins,  which  agglutinate  or  precipitate 
their  antigens  preparatory,  in  a  sense,  to  their  final  disintegration.  For 
still  more  complex  antigens  nature  has  provided  special  ferments,  al- 
ways present  in  varying  proportions  in  the  blood,  which,  when  united 
with  the  antigen,  cause  its  disintegration  and  solution  in  a  manner  simi- 
lar to  the  process  of  digestion  as  it  takes  place  in  the  intestinal  canal. 
These  ferments  are,  however,  powerless  unless  united  with  the  antigens, 
and  here  we  find  that  the  antibody  serves  as  the  connecting  link,  bind- 
iaguantige.il  with  ferment,  which  results  in  a  form  of  digestion  and  final 
lysis  or  solution.  The  antibody  is,  therefore,  simple  in  structure,  and 
is  composed  of  two  binding  or  grasping  arms — one  for  the  antigen  and 
one  for  a  ferment.  This  interbody,  or  amboceptor,  is  specific  for  the  anti- 
gen, and  will  act  only  and  specifically  with  this  antigen.  It  is  important 
to  remember  that  the  ferment  or  complement  is  not  an  integral  part  of 
the  antibody,  but  is  free  in  the  blood-stream;  that  the  antibody  is  only 
a  connecting  link,  but  preserves  its  importance  by  being  specific  for  its 

antigen;  that  the  primary  function  of  this  antibody  is  to  unite  antigen 

338 


AMBOCEPTORS   AND    COMPLEMENTS  339 

and  complement,  and  that  the  latter  then  causes  the  lysis  or  solution  of 
the  antigen.  The  connecting  link  or  antibody  of  this  nature  is  known 
as  an  antibody  or  receptor  of  the  third  order. 

Different  cells  produce  their  own  and  specific  interbodies  or  ambo- 
ceptors.  Thus  bacteria  or  vegetable  cells,  blood-corpuscles,  and  va- 
rious other  cells,  such  as  ciliated  epithelium,  spermatozoa,  renal  epi- 
thelium, etc.,  when  present  in  the  form  of  an  infection  or  when  injected 
into  an  animal,  generate  different  and  specific  amboceptors,  which  bring 
about  their  solution  by  binding  them  with  a  ferment  or  complement. 
One  ferment  or  complement  does  not  serve  for  all;  there  are  various 
ferments,  which  act  with  the  different  amboceptors,  but  all  have  proper- 
ties so  nearly  alike  that  many  believe,  with  Bordet,  that  but  a  single 
complement  exists. 

Definition. — This  special  digestive  and  lytic  process  is  known  to 
occur  with  cells,  and  hence  the  antibodies  capable  of  bringing  about  this 
action  are  called  cytolysinSj  or  substances  that  cause  lysis  or  solution  of 
the  various  cells  that  may  be  their  antigens. 

It  is  well  to  remember  that,  according  to  Ehrlich,  the  three  orders  of 
antibodies  each  have  their  counterpart,  both  in  structure  and  in  effect, 
in  the  receptors  serving  for  the  normal  nutrition  of  cells.  For  the  sim- 
plest molecule  of  food  that  is  in  solution  the  cell  is  provided  with  a  simple 
receptor  for  union  with  the  molecule,  which  is  then  directly  assimilated. 
This  receptor  is  similar  to  an  antitoxin,  or  an  antibody  of  the  first  order, 
which  destroys  its  toxin  directly  and  without  further  ado.  More 
complex  food  material  must  first  undergo  some  preparation  by  the  cell 
before  it  can  be  assimilated,  and  accordingly  we  find  receptors  provided 
with  a  more  complex  structure  which  have  their  counterpart  in  the  anti- 
bodies of  the  second  order,  or  those  possessing  a  special  toxic  portion 
that  agglutinates  or  precipitates  their  antigen  or  prepares  it  for  phagocy- 
tosis. It  is  possible  that  with  physiologic  substances  this  is  all  that  the 
cell  requires  of  its  receptor,  but  so  far  as  is  known,  it  would  appear  that 
for  antibodies  this  action  does  not  in  itself  injure  the  antigen,  but  is 
rather  one  step  toward  preparation  for  its  further  destruction.  Or- 
ganized and  complex  food  substances  must  be  digested  before  assimila- 
tion can  occur,  and  here  we  find  that  the  receptor  acts  as  a  link  in  binding 
the  food  molecule  to  a  ferment,  with  resulting  dissolution  and  assimila- 
tion of  the  products  of  solution.  These  are  called  receptors  of  the  third 
order,  and  have  their  counterpart  in  similar  antibodies, — the  cytoly- 
sins, — which  act  as  links  or  interbodies  between  antigen  and  a  comple- 
ment, the  latter  being  entirely  free  and  separate,  and  independent  of 
the  receptor  or  antibody  (interbody)  itself  (Fig.  95). 


340 


CYTOLYSINS 


Varieties  of  Cytolysins. — The  cytolysins  produced  by  bacteria  are 
known  as  bacteriolysins,  i.  e.,  antibodies  producing  disintegration  and 
lysis  of  bacteria.  The  cytolysins  known  as  hemolysins  cause  lysis  or 
hemolysis  of  the  erythrocytes.  Similar  cytolysins  may  be  formed  for 
practically  all  cells,  such  as  leukocytes,  epithelium,  liver,  kidney,  spleen, 
etc.,  and  to  these  the  general  name  cytotoxin  has  been  given;  thus  we 
have  leukotoxin,  hepatotoxin,  nephrotoxin,  neurotoxin,  etc.,  these 


FIG.  95. — FORMATION  OF  CYTOLYSINS  (HEMOLYSINS,  BACTERIOLYSINS,  CYTOTOXINS). 

The  central  white  area  represents  a  molecule  of  a  cell;  the  shaded  portion  repre- 
sents the  cell  itself;  the  surrounding  area  represents  the  body-fluids  about  the  cell. 

R,  Receptor  of  the  molecule  '(third  order) ;  R2,  overproduction  of  receptors,  which 
are  being  cast  off;  A,  a  cast-off  receptor  which  now  constitutes  the  antibody  or  am- 
boceptor;  C,  molecule  of  complement  free  in  the  body-cells  and  body-fluids;  A2 A4, 
amboceptors  in  combination  with  molecules  of  a  cell  (antigen)  and  a  complement; 
A3,  an  amboceptor  in  combination  with  a  molecule  of  a  cell.  The  cell  (antigen)  is 
now  said  to  be  sensitized.  Lysis  does  not  occur  because  a  complement  is  not  united. 

terms  being  more  nearly  correct  and  expressive  of  the  actual  mechanism 
by  which  their  action  is  produced. 

Nomenclature. — In  no  other  field  of  immunity  have  so  many  differ- 
ent names  been  applied  to  the  same  substances  as  have  been  applied  to 
this  order  of  antibodies.  This  confusion  of  terms,  added  to  the  various 
interpretations  placed  upon  their  significance,  has  rendered  the  subject 
incomprehensible  to  those  not  specially  interested. 

The  ferment-like  and  thermolabile  substances  present  in  all  serums 
and  actively  concerned  in  lytic  processes  have  been  given  the  name  of 


AMBOCEPTORS  341 

alexin  by  Bordet;  Metchnikoff  called  it  cytase,  and-Ehrlich  designated 
it  as  complement  because  in  the  conception  of  the  side-chain  theory  it 
completes  the  reaction  after  being  linked  with  the  antigen.  The  term 
"alexin"  was  first  applied  by  Buchner  to  the  germicidal  substance  found 
in  normal  serum.  We  now  know  that  Buchner  was  working  with  both 
amboceptor  and  complement,  although  Bordet  was  the  first  to  discover 
the  former,  Buchner  having  been  unconsciously  most  interested  in  the 
thermolabile  complement. 

To  the  antibody  itself  the  term  substance  sensibilisatrice  has  been 
applied  by  Bordet,  for  he  believes  that  this  antibody  sensitizes  or  pre- 
pares the  cell  for  the  action  of  the  alexin  or  complement.  The  following 
names  have  been  applied  to  the  antibody  by  various  observers :  fixateur, 
by  Metchnikoff;  preparator,  by  Mliller;  and  amboceptor,  interbody,  and 
immune  body  by  Ehrlich.  Of  these,  the  term  "amboceptor"  is  in  most 
general  use,  signifying  a  two-armed  body  that  unites  antigen  on  the  one 
hand,  with  a  complement  on  the  other. 

When  using  the  term  amboceptor,  care  should  be  used  to  designate 
its  specific  character;  thus,  for  example,  a  hemolytic  amboceptor  and  a 
bacteriolytic  amboceptor  mean  respectively  a  hemolysin  and  a  bacterio- 
lysin. 

It  is  common  practice  to  designate  an  amboceptor  according  to  the 
cell  for  which  it  has  a  special  affinity;  thus  antisheep  amboceptor  or 
hemolysin  means  an  amboceptor  for  sheep  cells,  the  prefix  "anti"  being 
affixed  because  it  is  destructive  for  those  cells. 


AMBOCEPTORS 

Although  antitoxins  have  received  considerable  study  from  a  thera- 
peutic standpoint,  probably  no  order  of  antibodies  has  been  given  more 
attention  than  the  cytolysins  have  received,  not  only  because  of  their 
vast  therapeutic  possibilities,  but  also  from  their  value  as  an' aid  to  diag- 
nosis. The  hemolysins  especially  have  been  utilized  in  making  the 
Wassermann  test  for  syphilis  and  similar  reactions,  the  very  nature  of  the 
phenomenon  offering  a  visible  and  fascinating  method  of  study. 

Since  the  general  structure,  formation,  and  action  of  the  various  am- 
boceptors,  such  as  the  bacteriolysins,  hemolysins,  and  other  cytolysins, 
are  essentially  similar,  the  general  character  of  amboceptors  may  be  here 
considered,  a  study  of  the  special  characteristics  of  each  being  reserved 
for  subsequent  chapters  on  the  more  important  members  of  the  group. 

Historic. — The  alexins  or  complements  were  first  discovered  through 
the  researches  of  Nuttall  and  Buchner  in  1889.  The  amboceptors  were, 


342  CYTOLYSINS 

of  course,  present  in  the  various  serums  with  which  these  observers  were 
working,  but  it  was  not  until  1895  that  Bordet  showed  quite  clearly  that 
two  substances  were  concerned  in  the  phenomena  of  bacteriolysis  and 
hemolysis.  At  this  time  he  demonstrated  that  the  alexin  or  comple- 
ment may  be  removed  from  a  serum  by  heating  it  to  55°  to  56°  C.,  and 
that  it  may  be  reactivated  by  the  addition  of  fresh  serum  from  another 
animal;  that  an  old  bacteriolytic  serum  cannot  produce  bacteriolysis 
unless  it  is  reactivated  by  a  fresh  normal  serum  or  is  placed  in  the  peri- 
toneal cavity  of  a  living  animal,  from  which  it  may  derive  the  thermola- 
bile  alexin.  In  other  words,  the  amboceptors  in  these  serums  with- 
stood the  effects  of  heating  and  age,  but  were  unable  to  produce  lysis 
without  the  aid  of  an  alexin  furnished  by  a  fresh  normal  serum. 

Structure  of  Amboceptors. — According  to  the  theory  of  Ehrlich,  an 
amboceptor  is  but  a  simple  interbody  furnished  with  two  haptophore  or 
grasping  portions.  One  haptophore  group  attaches  the  antibody  to  its 
antigen,  whatever  that  may  happen  to  be — bacterium,  erythrocyte, 
epithelial  cell,  e.tc.,  while  the  other  attaches  a  suitable  complement 
(Fig.  95).  The  first  is  called  the  cytophile  or  antigentophile  group,  and 
the  second,  the  complementophile  group.  The  amboceptor  is  specific 
in  the  sense  that  it  will  unite  only  with  its  antigen  or  other  very  closely 
related  body.  For  example,  when  a  rabbit  is  injected  with  sheep  cor- 
puscles an  amboceptor  is  formed  that  will  unite  only  with  sheep,  and 
not  with  human,  dog,  ox,  or  other  cells. 

As  will  be  shown  further  on,  Ehrlich  believes  that  many  different 
complements  may  be  present  in  a  serum,  whereas  Bordet  believes 
that  one  complement  exists  that  will  act  with  the  amboceptor,  whether 
this  is  bacteriolysin  or  hemolysin.  This  view  is  based  mainly  upon  the 
observation  that  the  complement  in  a  serum  may  be  absorbed  out  by 
furnishing  an  excess  of  either  bacteriolytic  or  hemolytic  amboceptors, 
the  one  variety  of  amboceptor  removing  all  the  complement  for  the 
other.  Although  the  results  of  experimental  work  would  seem  to  indi- 
cate that  Ehrlich's  belief  in  the  plurality  of  complements  is  correct,  and 
while  this  view  is  quite  generally  held,  conclusive  proof  regarding  this 
has  not  as  yet  been  furnished.  An  amboceptor  may  have  more  than 
one  complementophile  group,  and  may  bind  a  number  of  different  com- 
plements simultaneously  (polyceptor)  (Fig.  96).  Ehrlich  and  Morgen- 
roth  called  attention  to  this  possibility  when  they  stated:  " Finally,  it 
is  possible  that  an  immune  body,  besides  one  particular  cytophile  group, 
contains  two,  three,  or  more  complementophile  groups."  Later  Ehrlich 
and  Marshall  showed  that,  in  order  to  get  a  specific  lytic  effect,  it  was  not 
necessary  for  all  complements  to  become  active,  but  that  only  a  few 


AMBOCEPTORS  343 

are  necessary  in  any  single  instance  to  bring  about  effect.  These  com- 
plements are  termed  "  dominant  complements,"  the  remainder  being 
known  as  "  non-dominant  complements." 

Whether  amboceptors  can  undergo  degenerative  changes  and  lose 
their  cytophile  or  complementophile  groups  and  become  amboceptoids, 
just  as  toxoids  and  agglutinoids  are  formed,  is  still  doubtful.  Reasoning 
from  analogy  to  the  toxins  and  agglutinins,  it  is  probable  that  ambocep- 
toids may  be  produced  by  a  loss  of  the  complementophile  group,  the 
cytophile  portion  of  all  antibodies  being  more  stable;  such  ambocep- 
toids, by  uniting  with  their  antigens,  may  effectually  block  the  action 
of  an  amboceptor,  just  as  agglutinoids  prevent  agglutination. 

General  Properties  of  Amboceptors. — Amboceptors  are  fairly  re- 
sistant bodies,  withstanding  to  a  well-marked  degree  the  effects  of  heat, 
acids,    alkalis,    exposure,    and    drying.     A 
hemolytic  serum,  for  instance,  may  be  pre- 
served  in   a    sterile    condition    for    many 
months  and  show  but  slight  deterioration 
in  its  activity.     Such  a  serum  may  be  dried 
in  vacuo  or   on   suitable    filter-paper,   and 
preserve  its  activity  for  remarkable  inter-      FIG.  96.— THEORETIC  STRUC- 

i  f    x-  -xl,     u  v    I.J.  J  11  TURE  OF  A  POLYCEPTOR. 

vals  of  time,  with  but  slight  and  gradual  A>  ^ain  poyioli  of  am_ 

deterioration.     While  a  temperature  of  55°      boceptor  in  combination  with 

...     .  ,  a  cell;  C,  dominant  comple- 

C.    will    inactivate     complement    in    from      ment;  c,  lesser  complements. 

fifteen  to  thirty  minutes,  amboceptors  can 

tolerate  from  60°  to  65°  C.  for  an  hour  and  show  but  slight  depreciation 

in  activity. 

Mechanism  of  the  Action  of  Amboceptors. — An  amboceptor  or  anti- 
body of  the  third  order  is  believed  to  act  as  a  simple  chemical  interbody 
between  antigen  and  complement — i.  e.,  it  is  a  connecting  link  between 
the  two.  Complement  is,  therefore,  the  active  agent  in  lytic  processes, 
but  is  practically  powerless  to  act  upon  the  antigen  until  brought  into 
chemical  union  through  the  intervention  of  an  amboceptor.  Comple- 
ment is  present  in  normal  serums,  and  its  quantity  cannot  be  increased 
by  immunization.  Amboceptors  are  specific  for  their  antigen,  may  be 
present  in  small  amounts  in  a  normal  serum,  but  may  be  greatly  in- 
creased during  infection  or  as  the  result  of  artificial  immunization.  A 
hemolytic  amboceptor  for  sheep  corpuscles  is  specific  for  these,  and  will 
not  unite  with  the  corpuscles  of  any  other  animal.  A  bacteriolytic 
amboceptor  as  for  Bacillus  typhosus  will  unite  only  with  those  bacilli, 
and  to  a  lesser  extent  with  closely  related  bacilli,  such  as  the  paratyphoids 


344  CYTOLYSINS 

but  not  with  other  bacterial  species  or  other  cells.  As  to  the  specificity 
of  complement,  opinions  differ.  Ehrlich  believes  that  there  are  many 
complements,  as,  e.  g.,  one  for  a  particular  hemolytic  amboceptor,  an- 
other for  a  bacteriolytic  amboceptor,  and  so  on  through  the  extensive 
list  of  known  amboceptors.  Bordet,  on  the  other  hand,  believes  that 
there  is  but  one  complement,  which  will  act  with  any  amboceptor. 

Bordet's  conception  of  the  mechanism  of  this  order  of  antibodies  is 
also  different  from  that  of  Ehrlich.  Both  agree  that  the  antibody  pre- 
pares the  cell  for  the  lytic  action  of  a  complement,  but  Ehrlich  holds 
that  the  antibody  acts  as  a  simple  link  between  antigen  and  comple- 
ment, whereas  Bordet  believes  that  the  antibody  acts  as  a  mordant  or 
dye,  penetrating  its  antigen,  and  sensitizing  or  preparing  it  for  the  action 
of  the  complement.  For  this  reason  Bordet  calls  these  antibodies 
"  sensitizers,"  or  substances  sensibilisatrice.  The  term  "  sensitizing "  is 
now  in  general  use,  and  is  employed  especially  in  hemolytic  tests  when 
corpuscles  or  bacteria  are  mixed  with  their  amboceptors  to  effect  their 
union.  It  is  expressive  and  satisfactory,  and  may  be  used  without 
necessarily  subscribing  to  Bordet 's  view. 

Metchnikoff  believes  that  amboceptor  or  his  "fixator"  is  found  in 
the  leukocytes.  He  asserts  that  the  amount  of  fixator  or  amboceptor 
produced  is  proportional  to  the  amount  of  phagocytosis  and  phagolysis 
that  occur  during  the  absorption  of  the  antigen.  Metchnikoff  considers 
the  fixators  as  analogous  to  enterokinase,  and  he  believes  that,  like  the 
latter,  the  fixator  acts  as  an  accessory  digestive  ferment,  having  for  its 
object  the  linking  of  the  more  potent  ferment,  as  a  cytase  or  comple- 
ment, to  the  cell  or  molecule  to  be  digested. 

Formation  of  Amboceptors. — While  experimental  data  are  at  hand 
to  show  that  amboceptors  may  be  produced  by  local  tissues,  it  is  entirely 
probable  that  in  wide-spread  infection  or  as  the  result  of  artificial  im- 
munization there  is  general  cellular  activity  with  extensive  antibody 
formation.  The  spleen  and  hematopoietic  tissues  in  general  and  the 
mononuclear  leukocytes  are  regarded  by  many  as  being  particularly 
active  in  the  formation  of  hemolysins  and  bacteriolysins  (Pfeiffer  and 
Marx,  Deutsch,  Wassermann). 

As  has  been  stated,  Metchnikoff  believes  that  antibodies  of  the  class 
under  consideration  are  the  products  of  the  leukocytes,  thus  tending  to 
preserve  the  importance  of  the  phagocytic  theory.  While  there  is  little 
doubt  that  the  various  leukocytes,  endothelial  cells,  and  other  phag- 
ocytic cells  are  sources  of  amboceptor  production,  there  is  no  reason  for 
accepting  the  belief  that  their  formation  is  confined  strictly  to  these  cells. 


AMBOCEPTORS  345 

Specifitity  of  Amboceptors. — It  has  been  stated  elsewhere  in  this 
volume  that  amboceptors  are  highly  specific  bodies.  This  specificity  is 
not,  however,  absolute,  for  just  as  group  agglutinins  are  produced  by 
one  bacterium  for  closely  allied  species,  so  in  like  manner  experimental 
investigation  by  Ehrlich  and  Morgenroth,  von  Dungern,  and  others  has 
shown  that  immunization  of  an  animal  with  the  erythrocytes  of  another 
animal  would  produce  one  chief  hemolysin  for  these  cells  and  a  second- 
ary hemolysin  for  the  cells  of  another  animal.  For  example,  on  immuniz- 
ing a  rabbit  with  ox  blood  a  hemolytic  serum  was  obtained  that  was 
hemolytic  not  only  for  goat  blood,  but  also  for  ox  blood.  These  second- 
ary amboceptors  are  known  as  group  or  partial  immune  bodies.  Their 
production  may  readily  be  understood  when  it  is  remembered  that  the 
body-cells  are  conceived  as  being  provided  with  various  side-arms  for 
many  different  blood-cells,  bacteria,  etc.  Now,  if  the  erythrocytes  of 
the  goat  possess  receptors  not  only  for  the  particular  goat-blood  side- 
arms  of  the  body-cells,  but  also  for  the  ox-blood  side-arms,  both  sets  of 
side-arms  will  be  attacked  and  consequently  two  amboceptors  are 
formed — one,  the  main  one,  for  goat  corpuscles,  and  a  secondary  one 
for  ox  corpuscles.  Ehrlich  and  Morgenroth,  therefore,  claim  that  the 
immune  body  of  a  hemolytic  serum  is  composed  of  the  sum  of  the  partial 
immune  bodies,  which  correspond  to  the  individual  receptors  used  to 
confer  the  immunity.  Since  the  cells  of  various  animals  of  the  same  and 
of  different  species  vary  in  the  number  and  variety  of  side-arms  or  re- 
ceptors, which  are  not  present  in  another,  the  different  combining  group 
possessed  by  a  blood-cell  or  a  bacterium  will  not,  therefore,  find  fitting 
receptors  in  every  animal,  and  thus  there  may  be  a  different  variety  of 
partial  immune  bodies  in  two  animals.  This  would  lead  to  the  possi- 
bility of  the  occurrence  of  antibodies  for  the  same  blood-cell  or  bacterium, 
differing  from  one  another  in  the  partial  immune  bodies  of  which  they 
are  composed,  depending  on  the  variety  of  the  animals  used  in  preparing 
the  serum. 

This  view  is  directly  opposed  to  that  of  Metchnikoff  and  Besredka, 
who  believe  that  a  certain  immune  body  is  always  the  same,  no  matter 
what  species  of  animal  was  used  in  preparing  the  serum.  As  will  be 
pointed  out  further  on,  in  addition  to  theoretic  interest,  the  subject 
possesses  great  practical  importance,  for,  as  is  well  known,  most  cura- 
tive serums  are  best  prepared  with  many  different  strains  of  a  particu- 
lar microorganism  because  of  certain  differences  in  their  antigenic  prop- 
erties, and  if,  in  addition,  the  value  of  a  bacteriolytic  serum  depends 
upon  the  sum-total  of  the  immune  bodies,  it  may  be  advisable  to  secure 


346  CYTOLYSINS 

as  many  of  these  as  possible  by  preparing  the  serum  from  various  animals 
of  the  same  and  of  different  species. 

It  will  be  understood,  therefore,  that  the  specific  action  of  antibodies 
of  this  order  is  not  limited  to  the  cells  used  in  the  immunizing  process, 
but  extends  to  other  cells  that  have  receptors  in  common  with  these, 
a  condition  that  is  analogous  to  group  agglutinins  and  precipitins  for 
closely  allied  cells  and  bacteria  or  dissolved  albumins. 

NATURAL  OR  NATIVE  AMBOCEPTORS 

Difference  Between  a  Normal  and  a  Specific  Immune  Serum. — Just 
as  small  and  varying  amounts  of  native  agglutinins  and  antitoxins  may 
be  found  in  normal  serums,  so,  in  like  manner,  various  native  bacterio- 
lytic  and  hemolytic  amboceptors  may  be  found.  According  to  the  side- 
chain  theory,  these  various  amboceptors  are  normally  attached  to  body- 
cells,  hence  it  is  probable  that  a  few  are  being  continually  swept  off  into 
the  blood-stream.  In  some  instances  the  amount  of  a  natural  ambocep- 
tor  may  be  quite  high;  thus,  for  example,  many  human  serums  contain 
relatively  large  amounts  of  antisheep  hemolytic  amboceptor.  These 
natural  amboceptors  will  be  considered  more  fully  in  the  chapters  on 
Hemolysins  and  Bacteriolysins. 

The  difference  between  a  normal  and  an  immune  serum  lies  in  the 
fact  that  the  normal  serum  contains  a  number  of  amboceptors  in  small 
amounts,  whereas  the  immune  serum  contains  a  greatly  increased 
amount  of  at  least  one  amboceptor  for  a  particular  cell.  As  has  been 
shown  by  numerous  investigators,  this  difference  is  not  due  to  the 
complements,  as  these  are  not  increased  during  the  process  of  immuni- 
zation. Since  the  presence  of  an  amboceptor  cannot  be  demonstrated 
unless  complement  is  present,  in  testing  a  serum  for  an  amboceptor  we 
must  furnish  sufficient  complement  to  bring  out  the  maximum  activity 
of  the  amboceptor.  If  the  serum  of  a  rabbit  before  and  after  immuniza- 
tion is  titrated  with  sheep  erythrocytes,  it  may  be  found  that  the  im- 
mune serum  contains  from  a  hundred  to  many  thousand  times  the 
normal  quantity  of  antisheep  amboceptor. 

These  facts  bear  a  further  practical  relation  to  the  treatment  of 
infectious  diseases  with  bacteriolytic  serums.  Ordinarily,  when  we  in- 
ject an  immune  serum  we  furnish  but  one  bactericidal  substance,  namely, 
the  bacteriolytic  amboceptor,  and  no  complement  at  all.  If  the  pa- 
tient's complement  is  decreased  or  at  least  insufficient  to  activate  the 
amboceptor  furnished,  lysis  will  not  occur,  and  accordingly  an  increased 
therapeutic  effect  may  be  secured  by  the  injection  simultaneously  of 


AMBOCEPTORS  347 

an  immune  serum  and  a  fresh  normal  serum.  This  procedure  presents 
certain  difficulties,  and  the  subject  is  considered  more  fully  in  the  chap- 
ter on  Passive  Immunization. 

Anti-amboceptors. — Just  as  anti-agglutinins  and  antiprecipitins 
may  be  formed,  so  anti-amboceptors  may  be  produced  experimentally 
by  immunizing  an  animal  with  an  amboceptor-laden  serum.  An  anti- 
amboceptor  is  specific  for  the  amboceptor  that  caused  its  production, 
and  when  these  are  mixed,  the  activity  of  the  amboceptor  is  impeded  by 
the  anti-amboceptor,  which  unites  with  its  cytophilic  group.  It  is 
possible  that  old  erythrocytes  are  destroyed  by  an  autohemolysin  present 
hi  the  blood-stream  under  normal  conditions,  and  that  a  physiologic 
equilibrium  is  maintained  through  the  production  of  an  anti-amboceptor. 

QUANTITATIVE  ESTIMATION  OF  AMBOCEPTORS 

Titration  of  a  Hemolytic  Amboceptor. — The  quantity  of  amboceptor 
in  a  given  amount  of  serum  may  be  determined  by  titration.  If,  for 
instance,  a  rabbit  is  injected  with  sheep  corpuscles,  the  amount  of  hemo- 
lytic  amboceptor  for  these  cells  in  the  rabbit  serum  may  be  determined 
by  the  following  method  of  titration:  To  a  series  of  test-tubes  increasing 
amounts  of  the  rabbit  immune  serum  (heated  to  remove  complement) 
are  added,  with  a  constant  dose  of  sheep-cells  and  a  constant  dose  of 
fresh  guinea-pig  serum  to  furnish  complement.  After  a  suitable  period 
of  incubation  the  tube  showing  complete  hemolysis  would  contain  suf- 
ficient hemolytic  amboceptor,  i.  e.,  just  one  unit.  The  value  of  the 
immune  serum  may  then  be  expressed  by  stating  that  so  much  serum, 
as,  e.  g.,  0.001  c.c.,  contains  one  unit  of  amboceptor.  Of  course,  if  the 
amounts  of  complement  or  corpuscles  are  varied  the  unit  will  likewise 
vary.  In  order  to  establish  or  measure  the  content  of  hemolytic  am- 
boceptor a  constant  amount  of  corpuscles  and  complement  must  be 
used.  The  details  of  this  amboceptor  titration  are  given  in  the  chapter 
on  Hemolysins. 

Titration  of  a  Bacteriolytic  Amboceptor. — A  bacteriolytic  amboceptor 
may  be  measured  in  much  the  same  manner  as  a  hemolytic  amboceptor, 
although  less  accurately,  because  of  technical  difficulties.  If  a  standard 
and  fixed  dose  of  an  emulsion  of  living  bacteria  and  a  fixed  dose  of  com- 
plement are  mixed  with  varying  amounts  of  inactivated  immune  serum 
containing  amboceptors  for  these  bacteria,  the  amount  of  amboceptors 
present  may  be  determined  by  plating  the  mixture  and  estimating  the 
number  of  bacteria  that  have  been  killed.  Or  we  may  use  fixed  doses  of 
immune  serum  and  complement  with  varying  amounts  of  bacteria  and 


348  CYTOLYSINS 

obtain  an  approximate  estimate  of  the  content  in  bacteriolytic  ambo- 
ceptors  after  the  same  manner. 

The  technic  of  these  tests  is  given  in  the  chapter  on  Bacteriolysins. 


/  COMPLEMENTS 

J 

Historic. — As  early  as  1876  Landois  described  the  hemolytic  action 
of  fresh  blood-serum  upon  the  blood-corpuscles  of  animals  of  certain 
species.  Traube  and  others  observed  that  animals  could  withstand  the 
injections  of  relatively  large  amounts  of  septic  material,  but  it  was  not 
until  1886-90  that  Fodor,  Nuttall,  Buchner,  and  others  fully  established 
the  bactericidal  properties  of  fresh  blood-serum.  Buchner  demon- 
strated the  fact  that  the  active  principle  causing  bacteriolysis  or  hem- 
olysis  is  very  labile,  and  can  be  inactivated  by  a  temperature  of  55°  C., 
by  dialysis,  or  by  dilution  with  distilled  water.  He  designated  the 
active  principle  "alexin." 

Subsequently,  in  1899,  Bordet  found  that  the  alexin  of  Buchner  was 
composed  of  two  distinct  substances — one  a  sensitizing  substance,  which 
is  thermostabile,  and  a  second,  the  thermolabile  substance.  Somewhat 
later  (1899)  Ehrlich  and  Morgenroth  confirmed  these  observations,  but 
applied  the  name  "amboceptor"  to  the  sensitizing  substance  and 
"complement"  to  the  alexin.  These  terms  are  most  widely  employed 
at  the  present  time.  Bordet  adheres  to  the  term  alexin,  meaning  thereby 
the  thermolabile  principle,  and  does  not  use  it  in  the  original  sense  of 
Buchner,  which  included  both  the  sensitizing  substance  and  the  alexin. 
Metchnikoff's  cytases  are  practically  the  same  as  Ehrlich's  complement 
and  Bordet's  alexin. 

J  Definition. — Complement  [Lat.,  complementum,  that  which  completes] 
is  the  substance,  present  alike  in  normal  and  in  immune  serum,  which  is 
destroyed  by  heating  to  55°  C.,  and  which  acts  with  an  amboceptor  to  produce 


As  mentioned  in  the  discussion  on  amboceptors,  the  complement  is 
the  active  lytic  substance  concerned  in  the  phenomenon  of  cytolysis, 
but  is  powerless  until  united  with  the  cell,  corpuscle,  or  bacterium  by 
means  of  the  interbody  or  amboceptor. 

Structure  and  General  Properties  of  Complement. — Complement  is 
ordinarily  not  attached  to  the  body-cells  and  is  free  in  the  blood-serum. 
According  to  Ehrlich,  complement  is  simple  in  structure,  and  is  composed 
of  a  haptophore  portion  for  union  with  the  complementophile  hapto- 
phore  of  an  amboceptor,  and  a  second  toxic  or  lytic  portion,  called  the 


COMPLEMENTS  349 

cytophile  group.  In  other  words,  the  theoretic  structure  is  similar  to 
that  of  a  toxin,  although  the  function  and  action  of  the  two  are  quite 
different. 

By  heating  a  complement  serum  to  55°  C.  for  half  an  hour  the  cyto- 
phile portion  is  destroyed,  and  the  serum  is  now  said  to  be  inactivated, 
as  the  complement  is  no  longer  active.  If  the  complement  serum  is 
allowed  to  stand  at  ordinary  room  temperature  for  forty-eight  hours  or 
longer,  the  same  change  will  take  place.  The  cytophilic  or  active  por- 
tion of  the  complement  is,  therefore,  quite  unstable.  When  this  portion 
is  altered  or  lost,  the  substance  is  called  complementoid,  and  this  is  anal- 
ogous to  toxoids  and  agglutinoids. 

Since  complementoids  have  their  haptophore  groups  intact,  they  will 
unite  with  amboceptors  and  to  some  extent  prevent  lysis  by  blocking  the 
active  complement,  just  as  toxoids  unite  with  antitoxin  and  agglutin- 
oids with  their  antigens. 

Anticomplements  may  be  obtained  by  immunizing  suitable  animals 
with  serums  that  contain  complement  or  complementoid.  When  an 
inactivated  anticomplement  serum  is  mixed  with  the  homologous  com- 
plement, the  haptophores  of  the  latter  are  bound  by  means  of  the 
haptophbres  of  the  anticomplements.  A  proof  of  this  union  lies  in  the 
fact  that  a  complement  serum  that  has  been  treated  with  its  specific 
anticomplement  is  no  longer  able  to  activate  an  appropriate  amboceptor. 

According  to  Gay,  the  production  of  anticomplements  is  only  ap- 
parent; he  explains  the  loss  of  complement  activity  when  a  fresh  serum 
and  its  antiserum  are  mixed  as  due  to  the  absorption  of  complement  in 
the  precipitate  which  forms,  although  the  latter  may  be  invisible. 

Anticomplements  may  be  of  practical  importance  owing  to  the  forma- 
tion of  auto-anticomplements.  The  complements  must  exercise  an  im- 
portant function,  not  only  in  the  destruction  of  bacteria,  but  also  in  the 
digestion  and  solution  of  all  kinds  of  foreign  albuminous  bodies  that 
enter  the  organism.  As  was  shown  by  Wassermann,  anticomplements 
may  so  bind  up  their  complements  as  to  render  then*  host  much  less 
resistant  to  certain  infectious  diseases.  The  spontaneous  development 
of  auto-anticomplement  in  an  animal  has  never  been  demonstrated,  as 
there  are  no  receptors  in  an  organism  of  the  complements  of  the  same 
organism.  The  injection  of  the  serum  of  another  animal  containing 
complements  that  are  almost  identical  may,  however,  lead  to  the  forma- 
tion of  an  auto-anticomplement  in  the  serum  of  the  immunized  animal. 

The  extreme  lability  or  sensitivity  of  complements  to  heat,  exposure, 
adds,  alkalis,  etc.,  is  their  most  prominent  general  characteristic.  An 


350  CYTOLYSINS 

active  serum  is  one  containing  complement,  and  this  must,  under  ordi- 
nary circumstances,  be  a  fresh  serum.  On  heating  or  exposure  the 
serum  becomes  inactive.  An  inactivated  serum  may  be  reactivated  by 
the  addition  of  fresh  complement  serum.  These  terms  are  in  general 
use,  especially  in  testing  for  hemolytic  and  bacteriolytic  reactions. 

Origin  of  Complement. — Buchner  regarded  complement  as  a  true 
secretory  product  of  the  leukocytes,  and  Metchnikoff  also  maintains 
that  leukocytes  are  the  main  source,  the  complement  being  liberated 
upon  disintegration  of  the  white  cells.  There  is  considerable  experi- 
mental evidence  both  for  and  against  these  views,  but  at  the  present 
time  the  consensus  of  opinion  would  tend  to  regard  the  leukocytes  as 
an  important,  but  not  necessarily  the  sole,  source  of  complement  forma- 
tion. 

Complement  has  been  demonstrated  in  plasma,  where  its  presence 
is  probably  due  to  continual  disintegration  of  leukocytes  and  liberation 
of  complement  during  life.  It  is  probably  increased  to  a  slight  extent 
as  serum  is  left  in  contact  with  the  blood-clot,  indicating  that  disintegra- 
tion of  leukocytes  may  augment  the  complement  supply. 

The  liver  (Muller  and  Dick),  pancreas,  and  other  organs  have  been 
regarded  as  sources  of  complement  formation,  but  in  general  the  evi- 
dence points  to  the  leukocytes  as  the  chief  source  of  supply,  the  liver 
being  probably  concerned  through  its  activity  in  blood  destruction. 

Multiplicity  of  Complements. — Ordinarily,  a  fresh  serum,  such  as 
that  of  the  guinea-pig,  will  furnish  complement  for  either  bacteriolytic 
or  hemolytic  amboceptors,  and  the  question  arises  as  to  whether  one 
complement  unites  equally  well  with  all  amboceptors,  or  whether  several 
complements  are  present  in  one  serum  that  act  more  or  less  specifically 
with  different  amboceptors. 

Bordet  believes  that  only  one  complement  is  present,  and  bases  this 
opinion  mainly  on  the  fact  that  a  complement  that  can  be  shown  to 
activate  either  a  hemolytic  or  a  bacteriolytic  amboceptor  may  be 
absorbed  out  of  a  serum  by  furnishing  an  excess  of  either  amboceptor. 

Metchnikoff  maintains  that  there  are  two  cytases  or  complements, 
one  being  derived  from  macrophages  and  mainly  hemolytic,  and  the 
second  derived  from  microphages  and  chiefly  bacteriolytic. 

Ehrlich  and  Morgenroth,  Sachs,  Wassermann,  Wechsberg,  and  the 
German  school  in  general  believe  that  many  different  complements  are 
present  in  amounts  varying  with  the  different  serums.  These  observers 
have  sought  to  prove  this  experimentally,  and  while  the  evidence  is  not 
absolutely  convincing,  because  of  the  difficulty  of  working  with  sub- 


COMPLEMENTS  351 

stances  that  are  so  labile,  yet  the  doctrine  of  the  multiplicity  of  comple- 
ments is  quite  generally  accepted. 

(a)  By  digesting  20  c.c.  of  fresh  goat  serum  that  was  found  to  activate 
different  hemolytic  amboceptors  with  3  c.c.  of  a  10  per  cent,  solution  of 
papain  in  the  incubator  for  from  thirty  to  forty-five  minutes,  it  was 
found  that  the  complement  for  one  amboceptor  was  destroyed,  whereas 
those  remaining  were  left  intact  or  but  slightly  impaired. 

(6)  By  treating  10  c.c.  of  this  goat  serum  with  1  c.c.  of  a  7  per  cent, 
solution  of  soda  for  an  hour  it  was  found  that  some  complements  were 
destroyed  and  others  were  weakened. 

(c)  By  sensitizing  different  blood-cells  with  homologous  amboceptors 
and  adding  these  to  a  fresh  serum  for  short  and  varying  periods  of  time, 
some  complements  could  be  destroyed,  whereas  others  would  be  left 
behind  with  undiminished  or  but  slightly  decreased  activity.     Pro- 
longed exposure  would  remove  all  complements. 

(d)  As  was  previously  stated,  anticomplements  may  be  produced  by 
immunizing  an  animal  with  the  complement  of  an  animal  of  a  different 
species.     The  anticomplements  appear  to  be  specific  for  the  comple- 
ments responsible  for  their  production,  and  by  means  of  these  anticom- 
plements different  complements  may  be  demonstrated  in  one  serum. 
Since  the  formation  of  anticomplements  would  depend  upon  whether  or 
not  the  body-cells  of  the  immunized  animal  possess  suitable  receptors  for 
the  various  complements,  in  a  series  of  animals  it  may  be  found  that  one 
does  not  produce  anticomplements  for  all  the  complements  injected, 
a  finding  that  would  tend  to  support  the  theory  of  the  multiplicity  of 
complements.     In  addition,  Marshall  and  Morgenroth  actually  found 
in  ascitic  fluid  an  anticomplement  for  at  least  one  of  two  complements 
present  in  guinea-pig  serum. 

These  experiments  go  to  show  that  complements  differ  in  this  respect 
at  least :  that  not  all  have  identical  haptophores.  Whatever  differences 
between  complements  exist  must  be  slight;  probably  the  cytophilic 
group  of  all  are  alike.  At  present  the  subject  has  more  theoretic  than 
practical  importance.  In  the  various  diagnostic  reactions  guinea-pig 
serum  ordinarily  furnishes  the  complement  for  hemolysin,  bacteriolysin, 
or  other  cytolysins,  and  in  the  therapeutic  administration  of  bacterioly- 
tic  serums  we  are  compelled  in  any  case  to  depend  for  activation  of  the 
amboceptor  upon  the  natural  complement  in  the  patient's  serum. 

The  Nature  and  Action  of  Complement. — The  true  nature  of  the 
complements  is  unknown.  In  many  respects  they  bear  a  resemblance 
to  ferments,  and  certainly  the  part  they  play  in  the  processes  of  cytolysis 


352  CYTOLYSINS 

suggests  a  ferment-like  activity.  They  differ  from  true  prqteolytic  fer- 
ments, such  as  trypsin,  in  not  digesting  the  stroma  of  corpuscles,  al- 
though recent  work  by  Dick  would  seem  to  indicate  that  proteolysis 
actually  occurs,  a  process  that  increases  the  permeability  of  the  cell  and 
permits  the  escape  of  hemoglobin. 

On  the  other  hand,  it  is  possible  that  the  nature  and  action  of  com- 
plements may  be  placed  upon  a  chemical  basis.  Following  the  dis- 
covery of  the  hemolytic  power  of  cobra  venom  by  Flexner  and  Noguchi, 
a  power  they  ascribed  to  the  presence  of  an  amboceptor  in  the  venom 
acting  with  serum  complement,  Kyes  found  that  the  amboceptor  may 
be  activated  not  only  by  a  complement  in  the  blood-serum,  but  also  by 
some  constituent  of  the  red  blood-corpuscles  themselves.  This  last 
observer  speaks  of  the  latter  as  endocomplement,  i.  e.,  endocellular  com- 
plement. 

In  attempting  to  discover  the  nature  of  this  endocomplement  various 
substances  existing  normally  in  the  erythrocytes,  such  as  cholesterin  and 
lecithin,  were  obtained  in  a  pure  state  and  their  activating  powers  for 
cobra  amboceptors  tested.  These  investigations  showed  that  lecithin 
has  an  activating  power,  whereas  cholesterin  is  antihemolytic.  Al- 
though all  erythrocytes  contain  lecithin,  yet  all  are  not  equally  suscep- 
tible to  the  action  of  venom  amboceptors,  which  is  probably  due  to  the 
fact  that  the  lecithin  in  the  cells  of  some  animals  is  bound  to  other  cell 
constituents  in  a  loose  way  and  is  thus  available  as  complement.  In 
syphilitic  infection  the  lecithin  content  of  the  erythrocytes  is  actually 
diminished  or  in  some  manner  rendered  less  available,  so  that  the  in- 
hibition or  absence  of  venom  hemolysis  is  diagnostic  of  this  infection. 

Kyes  was  able  to  obtain  the  union  of  cobra  amboceptor  and  lecithin, 
forming  what  is  known  as  cobra  ledihid.  Although  lecithin  is  an  unstable 
substance  and  is  difficult  to  obtain  free  from  fatty  acids  and  soap,  there 
is  little  doubt  but  that  Kyes'  lecithid  is  a  phosphatid  compound  and  is 
actively  hemolytic  after  all  traces  of  fatty  acids  have  been  removed. 

The  next  important  observations  were  made  by  Noguchi,1  who  found 
that  soap  isolated  from  blood  and  various  tissues  possessed  active  he- 
molytic properties.  The  salts  of  the  fatty  acids,  and  particularly  of  oleic 
acid,  were  found  to  possess  similar  hemolytic  properties.  Pure  soluble 
oleates  mixed  with  serum  were  found  to  produce  compounds  possessing 
many  of  the  characteristics  of  true  complements:  (1)  They  are  inacti- 
vated by  heating  to  56°  C.  for  half  an  hour;  (2)  they  are  inactive  at  0° 
C.;  (3)  the  addition  of  acids,  alkalis,  and  yeast  renders  them  inactive. 
1  Proc.  Soc.  Exper.  Biol.  and  Med.,  1907,  4,  107;  Biochem.  Zeitschr.,  1907,  6. 


COMPLEMENTS  353 

Von  Liebermann,1  as  the  result  of  his  own  experiments,  came  to  practi- 
cally the  same  conclusions,  and  advanced  the  hypothesis  that  the  com- 
plements of  the  blood  are  to  be  sought  for  in  the  soaps  of  the  serum; 
that  these  soaps  are  united  with  serum  albumin,  and  are  inactive  until 
liberated  by  the  amboceptors,  when  they  become  actively  hemolytic. 

Further  than  this,  it  was  shown  that  oleic  acid  may  act  as  an  am- 
boceptor,  and  when  added  to  an  inactive  soap-albumin  combination,  it 
would  render  this  actively  hemolytic.  Von  Liebermann  and  Fenyvessy2 
have  shown  that  a  mixture  of  soap,  serum,  and  oleic  acid  possesses  a 
striking  resemblance  to  complements  and  amboceptors,  and  that  the 
amboceptor-complement  action  is  much  more  than  a  mere  linkage  of 
complement  to  antigen  by  means  of  an  amboceptor.  These  observers 
suggest  that  an  amboceptor  may  have  an  affinity  for  certain  constituents 
of  the  cell  or  bacterial  body,  and,  on  the  other  hand,  act  upon  the  com- 
plement and  separate  one  of  its  constituents,  which  then  breaks  up  the 
cell.  These  artificial  hemolysins,  however,  completely  dissolve  the 
stroma  of  the  corpuscles,  whereas  the  immune  hemolysins  appear  to 
dissolve  out  the  hemoglobin,  leaving  the  stroma  undissolved.  As  has 
been  mentioned  elsewhere,  recent  work  would  tend  to  show  that  the 
stroma  is  also  dissolved,  at  least  in  part,  in  specific  hemolysis,  so  that  the 
difference  in  action  between  the  two  is  not  quite  so  apparent. 

While  the  simplicity  of  the  substances  concerned  in  these  observations 
does  not  harmonize  with  the  great  variety  and  complexity  of  the  im- 
mune bodies,  nevertheless,  as  Adami  has  pointed  out,  the  points  of  re- 
semblance between  artificial  and  natural  complements  and  amboceptor 
are  so  striking  that  material  advances  in  our  knowledge  of  their  nature 
and  action  may  be  gained  by  further  researches  into  the  chemistry  of 
immunity. 

Complement  Splitting. — During  recent  years  considerable  attention 
has  been  directed  toward  a  phenomenon  known  as  the  splitting  of  com- 
plement. It  was  generally  conceded  that  when  treated  with  hydro- 
chloric acid  (Sachs),  carbon  dioxid  gas  (Liefmann),  or  acid  and  alkaline 
phosphates  (Michaelis  and  Skwissky),  or  when  dialyzed  against  dis- 
tilled water  (Ferrata),  complement  may  be  split  into  two  parts,  known  as 
a  mid-piece  and  an  end-piece.  According  to  certain  investigators,  these 
two  components  of  the  complement  differ  in  their  behavior  in  hemolytic 
processes:  one,  the  mid-piece,  is  bound  by  the  sensitized  cells,  while 
the  other,  or  end-piece,  possesses  the  lytic  action.  Speaking  in  terms 
of  the  side-chain  theory,  it  is  just  as  if  the  haptophore  and  cytophilic 

1  Biochem.  Zeitschr.,  1907,  4.  2  Biochem.  Zeitschr.,  1907,  5. 

23 


354  CYTOLYSINS 

portions  of  the  complement  were  separated:  the  haptophore  portion 
corresponding  to  the  mid-piece  (the  cell  or  bacterium  being  the  first 
piece),  and  the  lytic  cytophilic  portion  corresponding  to  the  end-piece. 
In  successful  complement  splitting  the  mid-piece  is  believed  to  be  in  the 
globulin  fraction  and  the  end-piece  in  the  albumin  fraction.  Noguchi 
and  Bronfenbrenner1  have  cast  considerable  doubt  upon  these  views, 
and  have  shown  that  what  is  known  as  complement  splitting  is  really 
nothing  more  than  an  inactivation  of  the  active  principle  of  complement, 
since  both  globulin  and  albumin  fractions  contain  a  part  of  the  comple- 
ment, a  fact  that  can  be  demonstrated  by  the  removal  of  the  inhibiting 
action  of  the  acid  or  alkali  used  in  the  process. 

THE  BORDET-GENGOU  PHENOMENON  OF  COMPLEMENT  FIXATION 

In  the  endeavor  to  demonstrate  the  unity  of  complement  Bordet 
and  Gengou  devised  an  experiment  that  has  proved  of  great  practical 
value  in  the  serum  diagnosis  of  syphilis  and  other  infectious  diseases. 
By  mixing  bacteria  and  their  amboceptors  with  a  little  fresh  serum  con- 
taining complement  and  letting  the  mixture  stand  aside  for  an  hour  or 
so  it  was  found  that,  upon  the  addition  of  corpuscles  and  their  ambo- 
ceptor,  hemolysis  did  not  occur,  although  the  serum  that  had  been  used 
as  complement  was  capable,  in  its  original  condition,  of  producing  hem- 
olysis of  these  corpuscles.  Bordet  advanced  this  experiment  to  show 
that  the  complement  concerned  in  bacteriolysis  is  the  same  as  that  at 
work  in  hemolysis,  and  consequently  concluded  that  there  is  but  one 
single  complement. 

This  experiment  of  Bordet  is  usually  spoken  of  as  the  "Bordet- 
Gengou  phenomenon,"  and  is  now  used  extensively  in  determining 
whether  or  not  a  given  serum  contains  certain  amboceptors.  The  serum 
to  be  tested  is  first  inactivated,  treated  with  the  antigen  composed  of  an 
emulsion  of  the  bacterium  whose  amboceptor  it  is  desired  to  discover, 
and  then  mixed  with  a  small  quantity  of  a  fresh  normal  complement 
serum.  The  mixture  is  placed  in  the  incubator  for  an  hour,  during  which 
time  the  bacterial  antigen  unites  with  its  amboceptor  and  the  comple- 
ment, i.  e.,  fixes  the  complement,  so  that  when  red  blood-cells  previously 
sensitized  with  heated  hemolytic  serum  are  added,  hemolysis  does  not 
occur  because  the  complement  in  the  fresh  serum,  which  was  suitable 
for  lysis  of  the  sensitized  corpuscles,  has  been  "fixed"  by  the  bacteria 
by  reason  of  the  presence  of  specific  amboceptors  in  the  serum  tested 

1  Jour.  Exper.  Med.,  1912,  15,  598  and  625. 


COMPLEMENTS 


355 


(Fig.  97).  If  these  amboceptors  were  not  present,  then  the  complement 
would  remain  unfixed  and  be  free  to  hemolyze  the  sensitized  corpuscles, 
a  negative  reaction  being  indicated,  therefore,  by  hemolysis,  whereas 
the  absence  or  inhibition  of  hemolysis  indicates  a  positive  reaction. 


•C.A.C. 


FIG.  97. — MECHANISM  OP  COMPLEMENT  FIXATION. 

Tube  1  shows  the  hemolytic  system;  C,  a  red  blood-corpuscle;  A,  a  hemolytic 
amboceptor;  Cp,  complement;  C.A.C.,  complement  united  to  a  corpuscle  by  means 
of  the  specific  amboceptor.  Hemolysis  results. 

Tube  2  shows  complement  fixation  by  bacterial  antigen  and  amboceptor;  Ai 
antigen;  C,  complement  united  to  the  antigen  A\  by  the  amboceptor  A.  When 
hemolytic  amboceptors  are  added  hemolysis  does  not  occur  because  the  complement 
has  been  previously  fixed  by  the  bacterial  antigen  and  amboceptor. 

Tube  3  shows  absence  of  complement  fixation  because  the  bacterial  amboceptor 
Ai  is  not  specific  for  the  bacterial  antigen  A2  and  hence  complement  is  not  fixed; 
when  hemolytic  amboceptor  and  the  corresponding  corpuscles  are  added  comple- 
ment unites  with  these,  C.A.C.  and  hemolysis  occurs. 


Wassermann,  Neisser,  and  Bruch  have  applied  this  test  to  the  serum 
diagnosis  of  syphilis,  the  technic  of  the  test  being  considered  in  a  subse- 
quent chapter. 

Deviation  or  Deflection  of  Complement. — While  large  doses  of  anti- 
toxin are  indicated  in  the  treatment  of  diphtheria  and  tetanus,  theoretic- 
ally the  administration  of  too  large  an  amount  of  a  bacteriolytic  serum 


356  CYTOLYSINS 

may  result  in  more  harm  than  good.  As  has  been  pointed  out  by  Neisser 
and  Wechsberg,  more  amboceptors  may  be  introduced  than  can  be  taken 
up  by  the  bacteria  causing  the  infection,  and  those  that  remain  free  are 
capable  of  combining  with  some  of  the  complement  that  is  present,  and 
thus  prevent  a  portion  of  the  complement  from  acting  with  the  ambo- 
ceptors attached  to  the  bacteria  —  i.  e.,  the  complement  has  been  de- 
viated or  deflected  from  its  natural  course.  This  would  really  mean  a 
decrease  in  bacteriolysis,  but  while  deviation  of  complement  has  been 
demonstrated  as  a  fact,  its  importance  in  serum  therapy  is  probably 
overrated  and  has  certainly  not  been  definitely  proved. 


QUANTITATIVE  TITRATION  OF 

Titration  of  Hemolytic  Complement.  —  The  quantity  of  a  hemolytic 
complement  present  in  a  fresh  serum  for  certain  corpuscles  may  be 
measured  in  the  same  manner  as  hemolytic  amboceptors  are  measured, 
namely,  by  adding  to  a  series  of  test-tubes  increasing  amounts  of  the 
fresh  serum  with  a  constant  dose  of  an  emulsion  of  corpuscles  and  a 
constant  and  sufficient  dose  of  the  corresponding  hemolytic  amboceptor 
for  these  corpuscles.  After  a  suitable  period  of  incubation,  that  tube 
that  shows  complete  hemolysis  contains  just  sufficient  complement,  or 
one  unit.  Since  we  know  how  much  complement  serum  was  placed  in 
this  tube,  we  now  know  that  this  quantity  contains  one  unit  of  hemolytic 
complement.  Of  course,  the  amount  of  corpuscles  and  amboceptor  must 
be  constant  in  all  tubes;  if  the  quantity  or  quality  of  one  or  both  of  these 
is  altered,  the  unit  of  complement  will  vary. 

Titration  of  a  Bacteriolytic  Complement.  —  The  quantity  of  bacterio- 
lytic  complement  in  a  given  amount  of  fresh  serum  may  be  determined 
in  a  similar  manner,  although  far  less  accurately,  for  instead  of  viewing 
the  results  of  lysis  in  the  test-tube  as  we  can  do  in  hemolysis,  the  degree 
of  lysis  must  be  determined  by  plating  out  the  mixtures  to  determine  the 
relative  numbers  of  living  and  dead  bacteria.  The  degree  of  bacterio- 
lysis may,  however,  be  viewed  after  a  manner  with  the  aid  of  the  micro- 
scope. 

Gay  and  Ayer  employ  a  direct  method,  which  consists  in  adding 
varying  amounts  of  the  serum  to  be  tested  to  a  definite  volume  (0.5  c.c.) 
of  a  suspension  of  cholera  vibrios,  prepared  by  emulsifying  a  twenty- 
four-hour  agar  culture  in  10  c.c.  of  normal  salt  solution  and  adding  a 
constant  and  sufficient  dose  of  serum  from  a  rabbit  immunized  against 
cholera.  The  mixtures  are  placed  in  small  test-tubes  and  incubated  for 
one  and  one-half  hours  at  37°  C.  Films  are  then  prepared,  stained,  and 


COMPLEMENTS  357 

examined  to  ascertain  the  degree  of  changes  undergone  by  the  vibrios. 
Or  the  changes  may  be  observed  in  hanging-drop  preparations.  In 
either  case  a  control  is  prepared  of  the  culture  which  has  also  been 
incubated  along  with  the  mixtures.  Gay  and  Ayer  found  that  0.02  c.c. 
of  normal  human  serum  contained  sufficient  complement  to  effect  com- 
plete lysis  of  this  dose  of  vibrios — even  0.001  c.c.  produced  distinct 
changes. 


CHAPTER  XIX 
BACTERIOLYSINS 

HAVING  considered  the  general  nature  and  properties  of  amboceptors 
and  complements  and  the  mechanism  of  their  action  in  producing  solu- 
tion or  lysis  of  cells,  we  will  now  study  more  closely  the  bacteriolysins, 
which  are  antibodies  belonging  to  this  group  and  possessing  diagnostic 
and  considerable  therapeutic  importance. 

Historic. — The  early  history  of  the  discovery  of  the  bacteriolysins 
is  closely  associated  with  the  history  of  immunity  in  general,  for  with 
the  discoveries  in  bacteriology  and  the  establishment  of  the  relation  of 
bacteria  to  disease,  it  followed  as  a  matter  of  course  that  investigations 
should  be  undertaken  to  ascertain  the  mechanism  of  resistance  to  and 
of  recovery  from  an  infection. 

In  1874  Traube  showed  that  sej^ifijiiaterial  may  be  destroyed  in  the 
blood  of  living  animals,  and  in  1881  Lister  demonstrated  the  same  phe- 
nomenon in  extravascular  blood.  These  experiments  were  naturally 
somewhat  crude,  as  they  antedated  the  period  in  which  the  pyogenic 
microorganisms  were  isolated  and  studied  in  pure  culture,  but  they 
served,  nevertheless,  to  demonstrate  the  germicidal  powers  of  the  blood. 

In  1886  Fodor  demonstrated  the  germicidal  action  of  blood-serum 
upon  anthrax  bacilli.  This  work  was  followed  shortly  after  by  that  of 
Flugge  and  Nuttall,  who  showed  the  germicidal  powers  of  the  body-fluids 
in  general  independent  of  cells;  The  controversy  between  the  adherents 
of  the  cellular  and  humoral  theories  of  immunity  now  began,  as  Metchni- 
koff  was  actively  engaged  in  studying  phagocytes  and  in  formulating 
his  phagocytic  theory. 

Buchner  and  others  took  up  the  subject,  emphasizing  the  important 
germicidal  powers  of  the  body-fluids  and  ascribing  this  function  to  the 
presence  of  "alexins"  (substances  that  ward  off  disease).  Buchner 
showed  that  if  the  serum  was  heated  this  germicidal  power  was  lost; 
hence  it  followed  that  the  active  bacteriolytic  agent  was  considered  very 
unstable  and  was  quickly  destroyed  outside  of  the  body. 

^n  1894  Pfeiffer  demonstrated  most  clearly  the  phenomenon  of  bac- 
teriolysis, which  gave  great  encouragement  and  impetus  to  studies  in 

358 


DEFINITION  359 

immunity,  and  incidentally  strengthened  the  claims  of  the  humoral 
theory.  He  showed  that  cholera  vibrios  introduced  into  the  peritoneal 
cavity  of  a  guinea-pig  that  had  been  immunized  against  cholera  lost 
their  motility  and  finally  became  disintegrated  and  passed  into  solution 
regardless  of  the  presence  of  cells. 

It  remained  for  Bordet,  however,  to  show  the  mechanism  of  this  in- 
teresting phenomenon.  This  observer  demonstrated  the  fact  that  the 
thermolabile  body  was  but  one  substance  concerned  in  the  reaction,  and 
that  the  specific  substance  was  thermostabile  and  the  actual  product 
of  immunization,  results  that  were  later  corroborated  and  elucidated  by 
his  researches  upon  hemolysis,  and  by  those  of  Ehrlich  and  his  pupils  on 
cytolytic  phenomena  in  general.  As  previously  mentioned,  Bordet 
retained  the  name  "alexin"  for  the  thermolabile  substance  and  applied 
the  new  term,  "  substance  sensibilisatrice,"  to  the  specific  thermostabile 
antibody.  Later,  both  substances  were  renamed  by  Ehrlich,  and  called 
" complement"  and  "amboceptor"  respectively. 

As  will  be  pointed  out  further  on,  as  the  result  of  these  observations 
Metchnikoff  modified  his  phagocytic  theory,  and  recognized  the  exist- 
ence of  both  substances,  which  he  named  "cytases"  and  "fixateurs," 
believing  that  both  were  derived  from  cells  classed  as  phagocytes. 

All  are  agreed  as  to  the  presence  of  two  different  bodies  in  the  body- 
fluids  concerned  in  bacteriolysis,  although  opinions  vary  as  regards  their 
origin  and  mechanism  of  action.  The  side-chain  theory  has  been  widely 
accepted  in  explanation  of  their  action,  and  the  terms  applied  by  Ehrlich 
to  the  two  substances  concerned,  namely,  complements  and  ambocep- 
tors,  are  in  general  use. 

Definition. — Bacteriolysins  are  substances  present  in  the  serum  and 
other  body-fluids  that  kill  bacteria  with  or  without  lysis. 

The  term  itself  would  infer  that  solution  or  lysis  of  the  bacterium  is 
an  essential  property  of  an  antibody  of  this  order.  Bactericidins  are 
substances  that  kill  bacteria  without  lysis,  and,  strictly  speaking,  an 
effort  should  be  made  to  differentiate  between  the  terms,  although  from 
a  practical  standpoint  this  is  not  important.  Certain  microorganisms 
may  be  killed  and  resist  solution  or  digestion  for  a  comparatively  long 
time,  whereas,  on  the  other  hand,  the  same  bacteria,  under  different 
circumstances,  may  readily  be  lysed. 

Although  the  endotoxins  liberated  from  the  lysed  bacteria  may  pro- 
duce symptoms  of  disease,  followed  by  death,  yet  the  bacterium  itself 
is  usually  destroyed  and  unable  to  proliferate.  A  bacteriolysin  is,  there- 
fore, always  bactericidal,  although  the  converse  is  not  necessarily  true. 


360  BACTERIOLYSINS 

Custom,  however,  has  never  strictly  differentiated  between  the  two 
terms,  and  bacteriolysis  appears  to  be  but  a  continuation  of  and  a  more 
nearly  complete  bactericidal  process.  Hence  the  definition  just  given 
covers  both  terms — bacteriolysins  and  bactericidins. 

Origin  of  Bacteriolysins. — Our  knowledge  regarding  the  origin  of  the 
bacteriolysins  is  quite  fragmentary.  The  investigations  of  Pfeiffer  and 
Marx  in  cholera,  and  Wassermann  in  typhoid,  have  shown  that  the 
spleen  and  hematopoietic  organs  in  general  may  be  especially  concerned. 

According  to  Metchnikoff,  the  "bacterial  fixateurs,"  which  are 
practically  the  bacteriolysins,  are  secretory  or  excretory  products  of 
phagocytic  cells,  especially  the  polynuclear  leukocytes  or  microphages. 
It  is  commonly  believed  that  during  infection  the  bacteria  cause  phag- 
olysis  or  disintegration  of  these  cells,  with  liberation  of  both  comple- 
ments (cytases)  and  amboceptors  (fixateurs),  which  produce  extracellu- 
lar lysis  of  the  invading  bacterium  (bacteriolysis).  If,  on  the  other 
hand,  the  phagocytes  are  fortified  and  phagolysis  is  prevented,  the  bac- 
teria are  phagocytized  and  undergo  intracellular  lysis,  a  condition  that, 
according  to  Metchnikoff,  may  be  induced  experimentally  by  giving  an 
animal  an  intraperitoneal  injection  of  sterile  bouillon  twenty-four  hours 
before  bacteria  or  other  cells  are  injected. 

That  leukocytes  afford  a  bacteriolytic  substance  is  supported  by  ob- 
servations showing  that  leukocytic  exudates,  secured  by  the  injection 
of  a  sterile  aleuronat  suspension,  possess  a  well-marked  germicidal  ac- 
tivity. Issaeff  found  that  the  intraperitoneal  injection  of  sterile  bouillon 
and  other  mild  irritants,  by  producing  a  leukocytic  exudate  that  supplied 
certain  bactericidal  substances  and  facilitated  phagocytosis,  increased 
the  resistance  of  animals  to  bacterial  infection.  At  one  time  surgeons 
made  practical  use  of  this  observation  by  injecting  nucleinic  acid  and 
other  substances  into  the  peritoneal  cavity  before  performing  laparot- 
omy,  in  order  to  induce  a  local  resistance  to  a  possible  infection. 


LEUKINS  AND  LEUKOCYTIC  EXTRACTS 

The  bactericidal  substance  contained  within  leukocytes  has  been  ex- 
tensively studied  by  Schattenfroh,  Schneider,  Peterson,  Hiss  and 
Zinsser,  Manwaring,  and  others.  It  has  been  observed  that  when  leu- 
kocytes are  suspended  in  diluted  blood-serum,  the  bactericidal  proper- 
ties of  the  serum  are  increased  without  coincident  destruction  of  the 
cells,  showing  that  the  leukocytes  may  secrete  germicidal  substances  into 
the  fluid.  The  same  observation  has  been  made  with  Bier's  congestive 


LEUKINS   AND    LEUKOCYTIC    EXTRACTS  361 

lymph,  indicating  that  this  activity  takes  place  both  in  the  test-tube 
and  in  living  tissues. 

Hiss,  and  later  Hiss  and  Zinsser,  found  that  autolyzed  leukocytic 
exudates  possess  some  bactericidal  activity,  and  that  they  may  pro- 
foundly modify  experimentally  induced  infection  of  rabbits  and  guinea- 
pigs  with  the  pneumococcus,  staphylococcus,  streptococcus,  and  other 
bacteria.  In  applying  this  method  of  treatment  to  man  by  means  of 
subcutaneous  injections,  these  investigators  observed  distinctly  bene- 
ficial results  in  cases  of  epidemic  cerebrospinal  meningitis,  lobar  pneu- 
monia, staphylococcus  infections,  and  erysipelas. 

Preparation  of  Leukocytic  Extracts. — Hiss  and  Zinsser  have  pre- 
pared leukocytic  extract  by  giving  rabbits  intrapleural  injections  of 
aleuronat  suspension.  Manwaring  has  secured  much  larger  quantities 
by  making  his  injections  into  the  horse. 

The  aleuronat  is  prepared  by  making  a  3  per  cent,  solution  of  starch 
in  bouillon  without  heating,  and  adding  5  per  cent,  of  powdered  aleuronat 
to  this  emulsion.  The  starch  helps  to  keep  the  aleuronat  in  suspension. 
The  mixture  is  boiled  for  five  minutes  and  placed  in  large  sterile  test- 
tubes,  20  c.c.  being  placed  in  each  tube.  Final  sterilization  is  done  in 
an  autoclave. 

For  making  the  injections  large  rabbits  are  selected.  The  hair  over 
both  sides  of  the  thorax  is  removed,  the  skin  is  sterilized  with  tincture 
of  iodin,  and  10  c.c.  of  the  aleuronat  suspension  are  injected  into  each 
pleura!  cavity  at  a  point  in  the  anterior  axillary  line,  at  the  level  of  the 
sternum,  great  care  being  taken  to  avoid  puncturing  the  lungs.  After 
twenty-four  hours  the  animals  are  chloroformed  and  the  pleural  cavities 
carefully  and  aseptically  opened.  The  cellular  exudate  is  pipeted  into 
centrifuge  tubes  containing  at  least  10  c.c.  of  sterile  1  per  cent,  sodium 
citrate  in  normal  salt  solution.  One  or  more  cubic  centimeters  of  exu- 
date may  be  obtained  from  each  cavity.  The  exudate  is  usually  tinged 
with  blood.  It  is  then  centrifuged  and  the  supernatant  fluid  removed. 
The  sediment  is  broken  up  with  a  platinum  spatula,  and  20  volumes  of 
sterile  distilled  water  are  added.  The  tubes  are  set  aside  in  the  incuba- 
tor for  twenty-four  hours,  after  which  cultures  are  made  to  insure  steril- 
ity. A  small  amount  of  preservative  may  be  added,  and  the  extract 
placed  in  bottles  or  ampules  ready  for  administration.  It  is  given  sub- 
cutaneously  in  doses  of  from  5  to  10  c.c.  every  four  to  six  hours. 

The  endocellular  bactericidal  substances,  or  endolysins,  mentioned 
in  a  previous  chapter,  which  can  be  extracted  from  leukocytes,  are  not 
in  the  nature  of  complements,  as  they  are  not  rendered  inactive  by  tern- 


362  BACTERIOLYSINS 

peratures  below  80°  C.  They  cannot  apparently  be  increased  by  im- 
munization, the  quantity  present  in  each  leukocyte  being  probably  at 
all  times  just  sufficient  for  the  digestion  of  a  limited  number  of  bacteria, 
which  can  be  ingested  at  one  time  by  the  individual  leukocyte.  It 
is  probable  that  the  excess  of  bactericidal  substance  is  thrown  off  into 
the  blood-stream,  representing  the  serum  bacteriolysins,  and.  at  least 
indicating  that  the  leukocytes  are  one  source  for  the  production  of  bac- 
teriolytic  amboceptors. 

Mechanism  of  Bacteriolysis.  —  According  to  the  side-chain  theory 
of  Ehrlich  a  bacteriolysin  is  an  antibody  of  the  third  order,  or  an  ambo- 
ceptor  furnished  with  two  haptophore  or  grasping  arms  for  uniting  the 
bacterium  on  one  side  with  a  suitable  complement  on  the  other.  The 
antibody,  therefore,  acts  simply  as  an  interbody  or  connecting  link; 
it  is  specific  for  the  bacterium  causing  its  production,  but  is  unable  itself 
directly  to  injure  the  bacterium,  lysis  being  brought  about  by  an  at- 

tached complement.  Bacteriolysis  is, 
therefore,  an  interaction  of  ambocep- 
-tor  and  complement  upon  the  bacterial 

FIG.    98.—  THEORETIC    STRUCTURE      b°dy  ^Flg*  9®' 

OF  A  BACTERIOLYTIC  AMBOCEP-  While    bactenolytic     amboceptors 

T.OR\  will  unite  with  their  antigens  under 

A,   Amboeeptor;     C,     comple- 
ment;    r,  receptor  of  bacillus;    h,      practically  all  conditions,  nevertheless 

a  suitable    ^^P^ent   may   not   be 


of  the  amboceptor.  present,  and  hence  a  bacteriolytic 

serum  may  not  be  active  in  all  animals. 

The  influence  of  bacteriolysins  upon  endotoxins  is  a  question  of  con- 
siderable interest  and  importance.  As  the  result  of  convincing  experi- 
ments performed,  especially  by  Pfeiffer,  it  is  evident  that  a  bacteriolytic 
serum  does  not  neutralize  the  endotoxin  at  the  time  the  bacterium  under- 
goes disintegration.  Highly  immune  serums  appear  to  be  unable  to  pro- 
tect an  animal  against  the  endotoxins,  and,  indeed,  may  even  increase  the 
intoxication  and,  by  liberating  an  excess  of  endotoxin,  kill  the  animal. 

The  bactericidal  substances  derived  from  leukocytes  are,  however, 
apparently  capable  of  neutralizing  endotoxins,  to  some  extent  at  least, 
as  Hiss  and  Zinsser  were  unable  to  ascribe  the  beneficial  effects  of  leu- 
kocytic  extracts  to  the  bacteriolytic  action  alone. 

As  mentioned  elsewhere,  both  Metchnikoff  and  Bordet  maintain 
that  the  bacteriolysin  is  in  the  nature  of  a  "sensitizer,"  preparing  the 
bacterium  for  the  action  of  the  alexin  or  cytase,  just  as  a  mordant  aids 
in  the  penetration  of  a  dye-stuff. 


LEUKINS   AND    LEUKOCYTIC    EXTRACTS  363 

General  Properties  of  Bacteriolysins. — As  with  other  cytolysins, 
the  bacteriolysins  are  thermostabile  and  resist  heating  to  60°  C.,  being 
gradually  destroyed  at  temperatures  ranging  from  70°  to  80°  C.  They 
are  likewise  highly  resistant  to  acids  and  alkalis,  and  when  preserved  in 
a  sterile  condition  with  the  addition  of  small  quantities  of  a  preservative 
bacteriolytic  serums  for  therapeutic  and  diagnostic  purposes  may  re- 
main active  for  long  periods  of  time.  Cholera  immune  serum  as  a  diag- 
nostic aid  in  making  the  agglutination  and  Pfeiffer  bacteriolytic  reac- 
tions is  best  preserved  in  dry  form,  the  serum  retaining  its  activity  under 
these  conditions  for  considerable  periods  of  time. 

Normal  Bacteriolysins. — As  a  result  of  the  contention  of  Metchnikoff 
that  bacteriolysins  (fixateurs)  are  produced  only  upon  the  disintegration 
of  leukocytes,  and  that  the  plasma  is  accordingly  free  from  these  anti- 
bodies, much  experimental  work  has  been  done.  The  weight  of  evi- 
dence is  against  this  view,  as  both  amboceptors  and  complements  have 
been  demonstrated  in  plasma,  although  the  quantity  of  bacterial  ambo- 
ceptors normally  present  in  the  body-fluids  is  quite  small.  It  is  quite 
natural  to  expect  that  under  normal  conditions  small  amounts 
will  be  present,  as  receptors  are  being  constantly  thrown  off  into  the 
blood,  and  leukocytes  are,  of  course,  being  constantly  formed  and 
destroyed. 

Specificity  of  Bacteriolysins. — The  bacteriolysins  are  highly  specific 
antibodies,  and  are  useful  in  making  the  differentiation  of  bacterial 
species.  Group  bacteriolytic  reactions  are  less  common  as  compared 
to  group  agglutination,  as  was  shown  by  Kolmer,  Williams,  and  Raiziss 
with  the  typhoid-colon  group  of  bacilli.  As  a  practical  procedure, 
however,  the  agglutination  reaction  is  so  easily  secured  as  to  be 
the  test  of  choice  in  making  a  differentiation  between  closely  allied 
organisms.  As  with  the  agglutinins,  the  influence  of  partial  bac- 
teriolysins may  be  removed  by  using  highly  immune  serums  in  high 
dilutions . 

Practical  Applications. — The  bacteriolysins  have  considerable  value 
in  the  following  procedures : 

1.  In  making  a  differentiation  of  bacteria,  especially  when  the  pres- 
ence of  cholera  vibrios  is  suspected. 

2.  In  the  diagnosis  of  certain  infections,  such  as  cholera  and  typhoid 
fever. 

3.  In  the  treatment  of  some  infections  with  specific  bacteriolytic 
serums. 


364  BACTEKIOLYSINS 

TECHNIC  OF  BACTERIOLYTIC  TESTS 
THE  PFEIFFER  EXPERIMENTS 

The  essentials  of  this  important  test  have  been  described  at  the  be- 
ginning of  this  chapter.  Briefly,  it  consists  in  making  intraperitongaL 
injections  of  a  bacteriolytic  serum  mixed  with  living  bacteria  into  a 
normaljguinea-pig.  The  resulting  bacteriolysis  is  studied  microscopically 
by  withdrawing  small  amounts  of  peritoneal  exudate  at  varying  inter- 
vals. By  performing  the  experiment  with  varying  dilutions  of  serum, 
the  bacteriolytic  titer  may  be  determined  by  noting  the  dilution  in  which 
bacteriolysis  just  fails  to  occur  in  a  specified  time. 

Pfeiffer  also  showed  that  the  phenomenon  could  be  produced  by  in- 
jecting a  mixture  of  serum  from  an  immunized  animal  and  the  culture 
of  cholera  into  the  peritoneum  of  a  normal  guinea-pig.  This  phenome- 
non appeared  when  an  old  specimen  of  serum  was  used,  as  well  as  when  a 
fresh  specimen  that  had  been  heated  to  60°  C.  was  employed.  Later, 
this  observer  found  that  if  an  old  immune  serum  was  injected  into  the 
peritoneal  cavity  and  allowed  to  remain  for  a  time,  it  regained  its  bac- 
tericidal powers. 

Pfeiffer  believed  that  the  bacteriolytic  substance  may  exist  in  the 
serum  of  jin  immunized  animal  either  in  an  active  or  in  an  inert  state. 
In  the  blood-serum  or  peritoneal  fluid  of  the  living  animal  it  occurs  as 
an  active  substance,  but  when  kept  for  a  few  days  or  when  heated 
rapidly  to  60°  C.  it  becomes  inert;  it  maybe  rendered  active  again 
by  coming  in  contact  with  the  lining  endothelial  cells  of  the  perito- 
neum. 

The  foregoing  constitutes  the  classic  Pfeiffer  experiment.  The  bac- 
teriolytic amboceptor  present  in  the  immune  serum  is  activated  by  the 
complement  furnished  by  the  guinea-pig..  The  same  serum  will  not 
produce  bacteriolysis  in  the  test-tube  in  case  it  has  been  heated  or  the 
complement  is  lost  through  age  unless  iresh  normal  serum  or  peritoneal 
exudate  is  added.  By  immunizing  guinea-pigs  with  gradually  increasing 
doses  of  cholera  serum  and  then  introducing  fatal  doses  of  cholera  cul- 
ture intraperitoneally,  the  same  phenomenon  of  bacteriolysis  is  observed. 
In  this  instance  the  guinea-pig  furnishes  both  amboceptor  and  comple- 
ment. 

By  these  and  similar  experiments  and  observations  Bordet  was  able 
to  show  the  role  played  by  the  two  bodies  concerned  in  cytplysis  in 
general,  namely,  the  vthermolabile  alexin  or  complement  and  the  specific 
sensitizer  or  bacteriolytic  amboceptor. 


TECHNIC    OF   BACTERIOLYTIC    TESTS  365 

Bacteriolytic  Test  in  vivo  for  the  Identification  of  Bacteria  Recovered 
from  Feces,  Water-supplies,  etc. — This  method  is  employed  chiefly  in 
the  identification  of  suspected  cholera  cultures.  According  to  Citron, 
in  Germany,  the  Pfeiffer  test,  made  with  microorganisms  obtained  in 
pure  culture  from  suspected  patients,  is  required  for  the  official  diagno- 
sis of  the  first  cases  of  cholera. 

As  a  rule,  the  agglutination  test  is  first  applied  in  making  the  diag- 
nosis of  a  suspected  microorganism,  as  the  technic  of  this  test  is  more 
easily  carried  out. 

This  bacteriolytic  test  may  also  be  employed  in  the  study  of  typhoid 
and  paratyphoid  bacilli,  although  bacteriolysis  of  these  microorganisms 
is  less  complete  than  that  observed  with  cholera,  and  agglutination 
tests  answer  all  practical  requirements. 

The  test  consists  in  mixing  varying  dilutions  of  a  known  and  highly 
immune  serum  with  a  constant  dose  of  unknown  microorganisms,  and 
injecting  the  mixtures  intraperitoneally  into  guinea-pigs.  After  twenty 
minutes  small  amounts  of  exudate  are  withdrawn  by  means  of  fine  capil- 
lary pipets,  and  studied  in  hanging-drop  preparations.  In  the  presence 
of  a  positive  reaction  the  bacilli  are  observed  to  lose  motility,  become 
swollen  and  coccoid  in  shape,  and  gradually  form  granules,  ultimately 
disappearing  in  complete  solution. 

Preparing  the  Immune  Serum. — A  highly  immune  serum  is  required. 
This  may  readily  be  prepared  by  giving  rabbits  a  series  of  intravenous 
injections  of  a  known  culture,  according  to  the  technic  described  in  the 
chapter  on  Active  Immunization  of  Animals.  The  official  test  in  Ger- 
many demands  that  the  serum  be  of  such  strength  that  0.0002  gram  of 
dried  serum  will  suffice  to  disintegrate  completely  within  one  hour  one 
loopful  (2  mg.)  of  an  eighteen-hour-old  culture  of  virulent  cholera  in 
1  c.c.  of  nutrient  bouillon  when  injected  into  the  peritoneal  cavity  of  a 
guinea-pig.  The  Hygienic  Laboratory1  in  Washington  is  prepared  to 
furnish  board  of  health  laboratories  with  a  dried  serum  of  high  titer  for 
diagnostic  purposes. 

In  testing  an  immune  serum  to  determine  its  bacteriolytic  titer  the 
dose  of  microorganisms  should  not  be  larger  than  one  loopful,  so  that  if 
any  particular  strain  of  cholera  or  typhoid  is  not  sufficiently  virulent, 
necessitating  the  use  of  larger  doses,  the  virulence  should  be  increased 
by  passing  the  organism  through  guinea-pigs. 

Method  of  Testing  the  Virulence  of  a  Culture. — The  unit  of  measure- 
ment is  a  2  millimeter  platinum  loop,  which,  when  loaded,  will  usually 
1  Personal  communication  from  Dr.  John  F.  Anderson. 


366  BACTERIOLYSINS 

hold  about  2  milligrams  of  microorganisms.  (See  p.  216.)  All  dilu- 
tions are  made  with  sterile  neutral  broth,  and  not  with  salt  solution. 
Mixtures  are  injected  intraperitoneally  into  250-gram  guinea-pigs  to 
determine  the  dose  that  will  be  fatal  within  twenty-four  hours. 

Great  care  should  be  exercised  in  all  manipulations  to  avoid  acci- 
dental infection.  The  mouth-ends  of  pipets  should  be  plugged  with 
cotton.  Sufficient  assistants  should  be  on  hand  to  facilitate  the  making 
of  injections  and  the  examination  of  peritoneal  exudates  with  ease, 
caution,  and  certainty.  All  pipets,  measuring  glasses,  test-tubes,  and 
hanging-drop  preparations  should  be  immersed  after  using  in  1  per  cent, 
formalin  solution  before  cleaning.  In  other  words,  every  precaution 
should  be  taken  to  carry  out  a  thorough  and  conscientious  bacteriologic 
Jechnic. 

Guinea-pig  No.  1:  Four  loopfuls  of  agar  culture  emulsified  in  4  c.c. 

of  bouillon;  inject  1  c.c.  intraperitoneally  (=1  loopful). 
Guinea-pig  No.  2:  1  c.c.  of  foregoing  emulsion  -f-  1  c.c.  of  bouillon; 

inject  1  c.c.  intraperitoneally  (  =  H  loopful). 
Guinea-pig  No.  3:  1  c.c.  of  first  emulsion  +  4  c.c.  of  bouillon;  inject 

1  c.c.  intraperitoneally  (  =  3-  loopful). 
Guinea-pig  No.  4:   1  c.c.  of  emulsion  No.  3  +  1  c.c.  of  bouillon; 

inject  1  c.c.  intraperitoneally  (  =  ^$  loopful). 

A  satisfactory  culture  is  one  in  which  a  dose  of  £  loopful  will  prove 
fatal  within  twenty-four  hours.  The  immune  serum  is  then  titrated  with 
five  times  this  amount  of  culture,  or  one  loopful. 

Method  of  Titrating  a  Bacteriolytic  Serum. — The  serum  is  inacti- 
vated by  heating  to  56°  C.  for  half  an  hour,  and  dilutions  are  made  with 
bouillon  in  sterile  shallow  glasses  or  test-tubes.  One  loopful  of  an  eigh- 
teen-hour  agar  culture  of  the  microorganism  is  thoroughly  emulsified—, 
jn  the  diluted  serum,  and  the  mixtures  are  injected  intraperitoneally 
in  250-gram  guinea-pigs.  Higher  dilutions  than  those  given  here  may 
be  employed  until  the  limit  of  bacteriolytic  activity  is  reached. 

1.  Mix  0.5  c.c.  of  inactivated  serum  with  4.5  c.c.  of  bouillon  (1  :  10). 
Place  2  c.c.  of  this  mixture  in  a  separate  test-tube,  and  add  2  loopfuls  of 
culture.     Inject  1  c.c.  (  =  0.1  c.c.  immune  serum). 

2.  Mix  2  c.c.  of  the  first  dilution  (1  : 10)  with  18  c.c.  of  bouillon  (  = 
1 : 100).     Place  2  c.c.  in  a  separate  tube  and  add  2  loopfuls  of  culture. 
Inject  1  c.c.  (  =  0.01  c.c.  immune  serum). 

3.  Mix  1  c.c.  of  the  second  dilution  (1  :  100)  with  4  c.c.  of  bouillon 
( =  1  :  500).     Place  2  c.c.  in  a  separate  tube  and  add  2  loopfuls  of  culture. 
Inject  1  c.c.  (  =  0.05  c.c.  immune  serum). 


TECHNIC    OF   BACTERIOLYTIC    TESTS  367 

4.  Mix  2  c.c.  of  the  third  dilution  (1  :  500)  with  2  c.c.  of  bouillon 
(=  1 : 1000).     Place  2  c.c.  in  a  separate  tube  and  add  2  loopfuls  of  cul- 
ture.    Inject  1  c.c.  (  =  0.001  c.c.  immune  serum). 

5.  To  1  c.c.  of  the  fourth  dilution  (1 :  1000)  add  9  c.c.  of  bouillon 
(=1 : 10,000).     Place  2  c.c.  in  a  separate  tube,  and  add  2  loopfuls  of 
culture.     Inject  1  c.c.  (  =  0.0001  c.c.  immune  serum). 

6.  Control:  Emulsify  2  loopfuls  of  culture  in  2  c.c.  of  bouillon.     In- 
ject 1  c.c.     This  animal  will  probably  succumb  within  twenty-four  hours. 

7.  Control:  To  2  c.c.  of  a  1  :  10  dilution  of  inactivated  normal  rabbit 
serum  add  2  loopfuls  of  culture  and  inject  1  c.c.  intraperitoneally.     If 
goats  or  horses  are  used  in  preparing  the  immune  serum,  this  control 
should  be  conducted  with  normal  goat  or  horse  serum.     According  to 
Kolle,  one  loopful  of  virulent  cholera  culture  is  destroyed  in  the  peri- 
toneal cavity  of  a  guinea-pig  by  0.1  to  0.3  c.c.  of  normal  rabbit's  serum; 
0.02  to  0.03  c.c.  of  normal  goat's  serum;    0.005  to  0.01  c.c.  of  normal 
horse's  serum. 

8.  Control:  A  pig  may  be  injected  with  1  c.c.  of  a  1  : 100  dilution  of 
the  immune  serum,  and  with  a  loopful  of  some  other  microorganism, 
such  as  Bacillus  coli.     This  control,  however,  is  not  absolutely  neces- 
sary. 

An  area  of  the  abdominal  wall  of  the  guinea-pig  about  one  inch  in 
diameter  is  shaved  and  cleansed  with  alcohol.  After  the  injections  have 
been  made  the  bacteriolytic  phenomena  are  observed. 

In  making  this  test  fine  capillary  pipets  are  prepared  and  used  for 
withdrawing  the  peritoneal  exudate. 

After  permitting  the  animal  to  inhale  a  few  drops  of  ether,  to  make 
sure  that  it  will  not  suffer,  a  small  incision  is  made  through  the  skin  of 
the  abdomen.  The  capillary  pipet,  the  large  end  of  which  is  kept  closed 
with  the  index  finger,  is  then  quickly  passed  into  the  abdominal  cavity. 
The  index-finger  is  released,  and  the  tube  is  gently  moved  about  and 
withdrawn.  As  the  result  of  capillary  attraction  sufficient  exudate 
usually  passes  into  the  tube  without  the  aid  of  suction  (Fig.  99).  The 
tube  may  be  fitted  with  a  rubber  teat  in  case  suction  should  be  necessary. 
Hanging-drop  and  smear  preparations  are  made  and  studied  microscop- 
ically (Fig.  100).  Stained  smears  are,  however,  less  reliable  and  not  so 
useful  in  determining  the  degree  of  bacteriolysis  (Fig.  102). 

It  is  best  to  withdraw  the  exudate  immediately  after  injection,  and 
then  at  intervals  of  ten,  twenty,  thirty,  forty,  and  sixty  minutes. 

A  hanging-drop  preparation,  made  from  the  culture  by  emulsifying 
a  minute  quantity  in  bouillon,  should  be  on  hand  as  a  control  in  studying 


368 


BACTERIOLYSINS 


bacteriolysis  in  the  exudate  (Fig.  101).  A  smear  of  the  culture  stained 
with  dilute  carbolfuchsin  should  also  be  on  hand  for  making  comparison 
with  smears  of  the  exudate  (Fig.  103). 

^JBacteriolysis  is  first  manifested  by  loss  of  motility.  As  the  process 
progresses  many  of  the  bacilli  become  swollen  and  distorted,  and  later 
irregular  and  broken  fragments  or  granules  become  apparent.  Finally, 
at  the  end  of  an  hour,  the  exudate  is  practically  sterile.  In  those  cases 
in  which  bacteriolysis  is  complete  in  an  hour  the  animal  generally  sur- 


FIG.  99..— REMOVING  EXUDATE  FROM  THE  PERITONEAL  CAVITY  OP  A  GUINEA-PIG 

(PFEIFFER  BACTERIOLYTIC  TEST). 

A  small  incision  is  made  through  the  skin,  and  the  exudate  is  withdrawn  by 

means  of  a  pipet. 

vives.  In  the  higher  dilutions  of  serum  bacteriolysis  is  incomplete,  and 
the  animal  becomes  toxic  and  may  succumb  after  twenty-four  hours, 
double  the  virulent  dose  being  used  in  all  injections. 

In  the  foregoing  titration  a  serum  that  is  bacteriolytic  in  dilutions 
of  1  :  1000  or  over  will  be  satisfactory  for  purposes  of  diagnosis.  When 
preserved  in  a  sterile  condition  in  separate  ampules  in  a  dark  cold  place 
the  titer  usually  remains  unaltered  for  several  months. 

Dried  Serum. — If  the  serum  is  dried  in  vacuo,  it  will  be  necessary  to 
titrate  with  dilutions  of  the  dried  products  in  bouillon,  as  during  the 


FIG.  102. — A  SMEAR  OF  PERITONEAL  EXUDATE  REMOVED  TWENTY  MINUTES  AFTER 

INJECTION  OF  CULTURE  OF  CHOLERA  IN  FIGS.  101  AND  103. 

Stained  with  1  :  10  carbolfuchsin. 


FIG.   103. — STAINED  PREPARATION  OF  CHOLERA  BEFORE  BACTERIOLYSIS. 


TECHNIC    OF   BACTERIOLYTIC   TESTS 


369 


process  of  drying  the  amboceptor  content  may  be  decreased.  Dried 
serum  is  to  be  preferred,  however,  as  it  keeps  much  longer  and  there  is 
no  danger  of  rendering  it  worthless  by  contamination.  After  drying, 
the  product  is  ground  in  a  mortar  and  stored  in  amounts  of  0.1  or  0.2 
gram  in  separate  ampules. 

In  determining  the  bacteriolytic  titer  of  dried  serum  0.1  gram  is 
carefully  weighed  out  and  dissolved  in  9.9  c.c.  of  sterile  bouillon.  From 
this  stock  dilution  other  dilutions  may  be  prepared,  in  the  manner  pre- 
viously described,  and  injected  with  a  loopful  of  the  culture. 


FIG.  100.— CULTURE  OF  CHOLERA  UN- 
DERGOING BACTERIOLYSIS.  A  POSI- 
TIVE PFEIFFER  REACTION. 

A  hanging  drop  of  peritoneal  exudate 
removed  from  a  guinea-pig  one-half  hour 
after  injection  with  1  c.c.  of  the  suspension 
shown  in  Fig.  101  with  1  c.c.  of  1 : 1000 
dilution  of  cholera  immune  serum.  Note 
that  the  bacilli  are  now  quite  short  and 
coccoid  in  shape.  At  the  end  of  seventy 
minutes  .the  exudate  was  sterile.  What 
appears  to  be  two  or  three  coccoid  forms 
in  apposition  is  really  one  bacillus  under- 
going lysis. 


FIG.  101. — CULTURE  OF  CHOLERA  BE- 
FORE BACTERIOLYSIS.     X  720. 
A  hanging  drop  of  cholera  in  normal 
salt  solution  prepared  from  a  twenty- 
four-hour  culture  of    cholera  on  agar- 
agar. 


After  securing  an  immune 
serum  of  satisfactory  potency  the 
main  test  may  be  conducted. 
While  the  technic  is  more  difficult 
than  that  of  agglutination  tests, 

the  results,  under  proper  conditions,  are  more  conclusive,  for  there  is 
less  likelihood  of  group  or  partial  amboceptor  activity  for  closely  allied 
bacteria  taking  place. 

Technic  of  the  Pfeiffer  Test. — The  suspected  culture  to  be  tested 
should  be  grown  on  agar  for  from  eighteen  to  twenty-four  hours,  and 
used  in  doses  of  one  loopful  (2  mg.). 

If  cholera  is  suspected,  cholera  immune  serum  of  known  titer  should 
be  used.     Dilutions  of  serum  are  made  with  sterile  bouillon,  and  2  c.c. 
24 


370  BACTERIOLYSINS 

of  each  dilution,  representing  2,  5,  and  10  times  the  titer  dose,  are  placed 
in  separate  glasses  or  small  test-tubes.  A  fourth  tube  should  contain  2 
c.c.  of  a  1  :  100  dilution  of  normal  serum,  according  to  the  animal  used 
in  producing  the  immune  serum;  a  fifth  tube  should  contain  2  c.c.  of 
sterile  bouillon  (culture  control). 

Two  loopfuls  of  suspected  culture  are  thoroughly  emulsified  in  each 
of  the  five  tubes  of  the  series,  and  1  c.c.  of  each  is  injected  intraperi- 
toneally  in  five  guinea-pigs  weighing  about  250  grams  each. 

At  intervals  of  ten,  twenty,  forty,  and  sixty  minutes  the  peritoneal 
exudate  should  be  removed  with  capillary  pipets  and  examined  in  hang- 
ing-drop preparations.  Smears  may  be  prepared  and  stained  with  dilute 
carbolfuchsin,  although  they  give  less  information  than  hanging-drop 
preparations. 

If  guinea-pigs  Nos.  1,  2,  and  3  show  granule  formation  at  the  latest 
after  an  hour,  while  in  the  fourth  and  fifth  animals  the  bacteria  remain 
whole,  motile,  and  well  preserved,  the  reaction  may  be  regarded  as 
positive  and  the  diagnosis  as  established. 

Pfeiffer  Bacteriolytic  Test  in  vivo  in  the  Diagnosis  of  Disease. — Bac- 
teriolysins  are  usually  produced  somewhat  later  than  agglutinins,  and 
reach  their  highest  point  of  production  during  convalescence.  Bac- 
teriolytic tests  are  used  only  for  diagnostic  purposes,  when  agglutination 
reactions  are  negative  or  doubtful.  The  most  satisfactory  results  from 
these  tests  are  obtained  in  cholera.  Bacteriolysis  with  typhoid  bacilli 
is  less  typical  and  more  incomplete;  with  the  paratyphoid  and  dysen- 
tery bacilli  it  is  even  more  unsatisfactory,  and  in  anthrax,  pest,  and  the 
various  diseases  due  to  cocci  it  does  not  occur. 

The  test  is  conducted  in  a  manner  similar  to  the  preceding  test,  ex- 
cept that  instead  of  an  immune  serum  the  patient's  serum  is  used,  with 
a  known  culture  of  typhoid,  cholera,  paratyphoid,  etc.,  according  to  the 
infection  suspected  to  be  present. 

One  cubic  centimeter  of  the  patient's  serum  is  secured  in  a  sterile 
manner,  inactivated  by  heating  to  55°  C.  for  one-half  hour,  and  dilutions 
of  1  :  20,  1  :  100,  1  :  250,  and  1  :  500  prepared  with  sterile  bouillon.  Two 
cubic  centimeters  of  these  dilutions  are  placed  in  separate  tubes,  and 
two  loopfuls  of  an  eighteen-hour  culture  of  the  test  organism  emulsified 
in  each.  A  fifth  tube  contains  2  c.c.  of  bouillon  with  two  loopfuls  of 
culture,  and  serves  as  the  culture  control. 

One  cubic  centimeter  of  each  dilution  and  of  the  culture  control  is  in- 
jected intraperitoneally  into  five  guinea-pigs,  and  the  exudate  examined 
after  twenty,  forty,  and  sixty  minutes. 


TECHNIC    OF   BACTERIOLYTIC    TESTS  371 

If  granule  formation  occurs  in  the  first  two  or  three  animals,  but  is 
absent  in  the  culture  control,  the  reaction  may  be  regarded  as  positive, 
and  the  diagnosis  of  typhoid,  cholera,  etc.,  considered  as  established. 

If  granule  formation  occurs  to  any  extent  in  the  fifth  animal  (culture 
control),  the  culture  is  to  be  regarded  as  unsuitable  and  the  test  repeated. 

Measuring  the  Bactericidal  Power  of  the  Blood  in  vitro  by  the  Plate 
Culture  Method  (Stern  and  Korte). — Since  the  earliest  days  of  bac- 
teriology the  aim  and  purpose  have  been  to  devise  some  means  by  which 
the  bactericidal  power  of  the  blood  could  be  measured,  just  as  is  done  in 
testing  an  ordinary  germicidal  substance,  namely,  by  adding  a  bacterial 
suspension  to  the  serum  and  observing  the  effect  on  the  bacterial  con- 
tent. This  is  shown  by  counting  the  bacteria  in  a  loopful  immediately 
after  adding  the  bacterial  suspension,  and  repeating  this  at  regular  in- 
tervals, the  counting  being  done  by  the  method  of  plate  culture. 

The  use  of  the  platinum  loop  in  these  tests  is  objectionable,  since  the 
dose  is  quite  variable,  depending  upon  whether  the  loopful  is  removed 
from  the  fluid  edgewise,  or  with  the  loop  held  flat,  like  a  spoon  in  use. 
If  the  serum  contains  agglutinins,  the  results  with  any  form  of  technic 
are  likely  to  be  irregular,  and  the  number  of  colonies  upon  the  plate  stand 
in  no  relation  to  the  actual  number  of  microorganisms  present.  The 
results  may  be  masked  by  a  rapid  multiplication  of  the  survivors,  and 
accordingly  several  controls  are  necessary  with  any  technic. 

Neisser  and  Wechsberg  have  recommended  the  so-called  bactericidal 
plate  culture  method.  By  this  method  the  patient's  serum  is  inactivated, 
and  varying  amounts  mixed  with  definite  and  constant  quantities  of 
bacteria.  To  this  a  constant  quantity  of  active  normal  serum  is  added 
as  complement,  to  reactivate  the  bacteriolytic  amboceptors.  These 
mixtures  are  then  incubated  for  several  hours.  To  determine  whether 
and  in  what  proportion  death  of  bacteria  has  resulted  from  the  effect  of 
the  bacteriolysins,  agar  is  added,  and  the  mixtures  are  then  plated  and 
incubated  for  twenty-four  hours  or  longer.  The  number  of  colonies  are 
then  counted  and  the  results  compared  with  control  plates  of  the  culture 
alone. 

Stern  and  Korte  have  modified  this  technic  slightly,  and  recommend 
the  procedure  as  a  substitute  for  the  Pfeiffer  test  in  the  clinical  diagnosis 
of  typhoid  fever.  The  method  spares  a  certain  number  of  animals,  but 
it  is  somewhat  more  complicated  than  the  Pfeiffer  reaction,  and  its  re- 
sults are  less  trustworthy.  As  a  clinical  test,  therefore,  it  is  to  be  recom- 
mended only  in  cases  in  which  the  agglutination  reactions  have  yielded 
uncertain  results,  although  with  practice  and  care  the  test  oftentimes 


372  BACTERIOLYSINS 

yields  uniform  and  satisfactory  results,  and  may  be  employed  in  making 
special  investigations  for  determining  the  bactericidal  power  of  the  blood, 
as  after  typhoid  immunization. 

Technic  of  the  Test. — The  requisites  for  success  are  that  all  vessels 
and  diluting  fluids,  as  well  as  the  serums  employed,  should  be  absolutely 
sterile.  To  secure  uniform  and  reliable  results  it  is  necessary  to  famil- 
iarize oneself  with  the  technic  by  repeated  practice.  In  order  to  carry 
out  the  steps  in  the  technic  according  to  strict  aseptic  bacteriologic 
principles  the  services  of  an  assistant  are  required. 

Sterile  bouillon  is  largely  used  throughout,  to  maintain  proper  os- 
motic conditions. 

With  so  many  tubes  and  controls,  it  is  important  that  all  tubes  and 
Petri  dishes  be  properly  labeled  with  a  wax-pencil  to  avoid  confusion. 

One  cubic  centimeter  of  sterile  patient's  serum  and  an  equal  amount 
from  a  normal  and  healthy  person  to  serve  as  a  control  are  inactivated 
by  heating  to  55°  or  56°  C.  for  half  an  hour.  These  serums  are  then 
diluted  1  :  50  by  adding  49  c.c.  of  sterile  salt  solution  to  each  and  mixing 
thoroughly. 

Complement  is  prepared  by  securing  4  to  5  c.c.  of  sterile  rabbit's 
blood  and  separating  the  serum.  This  serum  is  chosen  because  it  should 
be  from  the  same  species  of  animal  as  that  used  in  producing  the  immune 
serum  to  be  tested.  In  determining  the  bactericidal  titer  of  human 
serum  either  guinea-pig  or  rabbit  complement  serum  may  be  used. 
Dilute  2  c.c.  of  this  fresh  serum  (not  over  eighteen  hours  old)  with  18 
c.c.  of  sterile  normal  salt  solution  (1  :  10).  Dose,  0.5  c.c. 

Secure  a  good  twenty-four-hour  bouillon  culture  of  typhoid  bacilli 
(grown  in  the  incubator)  and  dilute  1  :  500  by  thoroughly  mixing  0.1 
c.c.  of  the  culture  in  a  flask  containing  50  c.c.  of  sterile  normal  salt  solu- 
tion. This  dilution  of  culture,  when  used  in  constant  doses  of  0.5  c.c., 
generally  yields  satisfactory  results,  but  it  may  be  necessary  to  try  it 
out  beforehand,  for  the  control  plates  must  regularly  show  a  uniformly 
good  growth  and  contain  many  thousands  of  colonies  before  uniform 
results  can  be  expected.  The  bactericidal  effect  will  then  be  distinctly 
shown  by  the  reduction,  in  the  proper  plates,  of  this  large  number  of 
colonies  to  zero  or  near  it. 

Place  1  c.c.  of  sterile  neutral  bouillon  in  each  of  12  test-tubes  which 
have  been  plugged  with  cotton,  sterilized,  and  large  enough  to  hold  at 
least  from  12  to  15  c.c.  Place  in  the  first  of  these  tubes  1  c.c.  of  the 
diluted  patient's  serum  and  mix  thoroughly  by  alternate  sucking  up  and 
forcing  out  of  the  fluid;  then,  with  the  same  pipet,  draw  up  1  c.c.  and 


TECHNIC    OF   BACTERIOLYTIC   TESTS  373 

transfer  it  to  the  second  tube  of  the  series;  mix  as  before,  and  transfer 
1  c.c.  to  the  third  tube;  continue  in  this  manner  to  the  last  tube, 
from  which,  finally,  1  c.c.  is  discarded. 

Each  tube  now  contains  1  c.c.  of  fluid,  representing  dilutions  of  1  :  100, 
1 : 200,  1  : 400,  1  :  800,  1  :  1600,  1  :  3200,  and  so  on  up  to  1  :  204,800  of 
the  patient's  serum.  Higher  or  lower  dilutions  than  these  may  be  em- 
ployed. It  is  very  desirable  that  the  dilutions  be  high  enough  to  secure 
the  limit  of  bactericidal  activity,  so  that  the  last  plates  will  show  an  in- 
crease in  the  number  of  colonies. 

Arrange  four  tubes  with  the  normal  control  serum,  each  containing 
1  c.c.  of  fluid  and  representing  the  first  four  dilutions,  viz.,  1  :  100, 
1 :  200,  1  :  400,  and  1  :  800. 

Label  each  tube  carefully  with  the  initials  of  the  patient  and  the  dilu- 
tion it  contains.  Add  0.5  c.c.  of  the  diluted  bacterial  emulsion,  and 
finally  0.5  c.c.  of  the  diluted  complement  serum,  to  each  tube. 

All  manipulations  should  be  made  with  strict  bacteriologic  care  and 
with  the  aid  of  an  assistant,  as  the  introduction  of  contaminating  micro- 
organisms that  may  cause  spore-formation  will  considerably  vitiate  the 
value  of  the  test. 

The  following  controls  are  necessary: 

1.  0.5  c.c.  culture  directly  in  a  sterile  Petri  dish  +  5  to  10  c.c.  of 

neutral  agar  cooled  to  42°  C.  and  thoroughly  mixed.  This 
control  will  show  the  original  number  of  bacteria  contained  in 
the  emulsion.  Mark  as  control  No.  1. 

2.  0.5   c.c.   culture  +  1.5  c.c.  bouillon.     Mark  as  control  No.  2. 

After  three  hours'  incubation  this  tube  receives  the  usual 
amount  of  agar  and  is  plated.  It  will  show  to  what  degree  the 
culture  has  multiplied  in  the  incubator. 

3.  Since  the  complement  serum  frequently  contains  bacteriolysin, 

it  is  necessary  to  control  this  element  carefully.  To  a  series  of 
four  tubes  add  the  following  doses  of  the  serum,  diluted  1  :  10: 
1  c.c.,  0.5  c.c.,  0.2  c.c. ,0.1  c.c.  Add  0.5  c.c.  culture  to  each 
tube,  and  sufficient  bouillon  to  make  the  total  volume  in  each 
tube  2  c.c. 

4.  0.5  c.c.  complement  +  1.5  c.c.  bouillon.    To  control  the  sterility 

of  the  complement  serum. 

5.  1  c.c.  of  patient's  serum  (1  : 100)  -f-  1  c.c.  bouillon.     To  control 

the  sterility  of  this  serum. 

6.  1  c.c.  of  the  control  serum  (1  :  100)  +  1  c.c.  of  salt  solution.    To 

control  the  sterility  of  this  serum. 


374  BACTERIOLYSINS 

7.  1  c.c.  of  the  patient's  serum  in  dilution  of  1 : 100  +  0.5  c.c.  cul- 

ture +  0.5  c.c.  bouillon.  A  control  on  the  possible  presence  of 
complement  in  this  serum. 

8.  1  c.c.  of  the  control  serum  in  dilution  of  1  :  100  +  0.5  c.c.  culture 

+  0.5  c.c.  bouillon.  A  control  on  the  possible  presence  of 
complement. 

All  tubes  with  the  exception  of  the  first  control  which  has  been  plated 
are  placed  in  an  incubator  at  37°  C.  for  three  hours.  At  the  end  of  this 
time  the  contents  of  each  tube  are  plated  in  neutral  agar.  Sterile  Petri 
dishes  should  be  properly  and  plainly  labeled  with  a  wax-pencil  and 
arranged  in  order.  A  flask  of  plain  neutral  agar  is  melted  in  boiling 
water  and  cooled  to  42°  C.  The  tubes  are  removed  from  the  incubator 
and  shaken  gently  and  with  the  aid  of  an  assistant  from  5  to  8  c.c.  of 
agar  are  added  carefully  to  each  tube  with  a  sterile  10  c.c.  pipet.  The 
contents  are  mixed  by  gentle  rotation  of  the  tube  and  then  poured  in  the 
corresponding  Petri  dish,  followed  by  an  additional  mixing  according  to 
the  usual  bacteriologic  procedure.  With  ordinary  speed  the  whole  set 
of  tubes  may  be  poured  in  a  satisfactory  manner  before  the  flask  of  agar 
has  had  time  to  harden. 

Another  method  of  plating  consists  in  pipeting  the  contents  of  each 
tube  into  its  corresponding  dish,  and  then  washing  the  tube  with  an 
additional  1  c.c.  of  sterile  salt  solution,  to  remove  all  traces  of  serum  and 
culture.  Or  the  end  of  each  tube  may  be  flamed  and  the  contents 
poured  directly  into  a  dish.  If  this  method  is  adopted,  small  test-tubes 
should  be  used.  While  the  method  is  more  convenient,  it  is  usually  not 
so  accurate  as  the  first  two  methods. 

Neisser  plates  but  5  or  10  drops  from  each  tube.  The  dose  decided 
upon  is  the  one  employed  throughout.  For  example,  it  would  be  erro- 
neous to  take  5  drops  from  one  tube  and  10  from  another.  Neisser, 
however,  uses  much  smaller  amounts  of  the  serum,  as  1,  0.3,  0.1,  0.03, 
and  0.01  c.c.,  instead  of  the  much  higher  dilutions  given  in  this  technic. 
These  differences  must  be  remembered  and  the  proper  dilutions  employed, 
and  but  5  to  10  drops  should  be  plated  in  performing  the  bactericidal 
plate  test  according  to  Neisser 's  technic. 

Topfer  and  Jaffe  pour  a  thin  layer  of  agar  into  a  Petri  dish  and  allow 
it  to  harden.  Upon  this  they  pour  the  culture-serum  agar  mixture, 
which,  after  settling,  is  covered  with  another  thin  layer  of  agar.  In 
this  way  a  culture  in  the  water  of  condensation  is  avoided.  In  the  usual 
technic  this  may  be  avoided  by  turning  the  plates  over  soon  after  harden- 
ing occurs  so  that  the  water  of  condensation  collects  on  the  cover. 


TECHNIC    OF   BACTERIOLYTIC    TESTS 


375 


All  plates  are  incubated  at  37°  C.,  for  from  twenty-four  to  thirty-six 
hours  and  the  colonies  then  counted.  In  some  plates  the  number  may 
be  so  large  that  counting  will  be  inaccurate  and  unnecessary.  Sig- 
nificance can  be  attached  only  to  marked  and  easily  recognizable  dif- 
ferences. According  to  Neisser,  the  growth  is  best  and  most  rapidly 
described  by  means  of  approximate  estimates,  using  a  scheme  somewhat 
like  the  following:  0  or  almost  none;  about  100;  several  hundreds; 
thousands;  very  many  thousands;  infinite  numbers.  A  distinct  bac- 
tericidal action  is  then  present  only  if  the  controls  react  normally,  and 
if  a  reduction  of  colonies  from  an  infinite  number  or  many  thousands  to  0 
or  very  few  has  occurred.  As  previously  stated,  the  test  can  then  be  re- 
garded as  satisfactory  only  if  the  lower  limits  of  bactericidal  activity  have 
been  reached  and  the  last  plates  show  an  increase  in  the  number  of  colonies. 
Examination  of  these  plates  is  very  much  facilitated  by  using  a  colony 
counter,  that  of  Stewart  being  particularly  serviceable  with  agar  plates. 

The  controls  are  first  examined  and  the  results  recorded,  and  finally 
those  of  the  patient's  serum  are  set  down. 

As  a  practical  illustration,  the  results  of  a  test  with  a  rabbit  typhoid 
immune  serum  are  given,  because  this  is  in  general  an  average  example 
of  those  usually  obtained: 


1  c.c.  DILUTIONS 
OF  IMMUNE 
SEBUM 

TWENTY-FOUR- 
HOUR  CULTURE 
OF  BACILLUS 
TYPHOSUS  (DILU- 
TION, 1:500),  C.c. 

RABBIT   COMPLE- 
MENT SERUM, 
1  :  10,  C.c. 

PLATES  POURED  AFTER  THREE  HOURS 
AT  37°  C.  COUNTED  AFTER 
TWENTY-FOUR  HOURS 

Immune  Serum 
(Inactivated) 

Normal  Rabbit 
Serum  (In- 
activated) 

1-100 

0.5 

0.5 

0.5 
0.5 
0.5 
0.5 

0.5 
0.5 

0.5 
0.5 
0.5 
0.5 

0.5 
0.5 

0.5 
0.5 
0.5 
0.5 

0.5 
0.5 

0.5 
0.5 
0.5 
0.5 

About  500  colo- 
nies 
About  125  colo- 
nies 
Sterile 
Sterile 
Sterile 
Two  colonies; 
practically 
sterile 
100  colonies 
About  800  colo- 
nies 
About  2000  col- 
onies 
Many  thous- 
and 
Many  thous- 
and 
Many  thous- 
and 

Many  thousand 
Many  thousand 

Many  thousand 
Many  thousand 

1:200  
1  :  400           

1  :  800  

1:1600  
1:3200  

1:6400  . 
1  :  12,800 

1:25,600  
1:51,200  

1:102,400  
1:204,800  

376  BACTERIOLYSINS 

CONTROLS 
Control  1:  0.5  c.c.  culture  plated  immediately:    many  thousands 

of  colonies. 
Control  2:  0.5  c.c.  culture  plated  after  being  incubated  three  hours: 

plate  very  crowded;   number  of  bacteria  increased. 
Control  3:   Varying  amounts  of  normal  rabbit  serum  used  as  com- 
plement.    The  first  plate  containing  0.1  c.c.  serum  showed  a 
slight  bactericidal  action;   the  remaining  plates  showed  many 
thousand  of  colonies  and  were  comparable  to  control  No.  1. 
Control  4:  0.5  c.c.  complement  serum:  sterile. 
Control  5:   0.01  c.c.  inactivated  immune  serum:   sterile. 
Control  6:  0.01  c.c.  inactivated  normal  control  serum:  plate  shows 

several  colonies  of  contaminating  bacteria. 
Control  7:   0.01  c.c.  inactivated  immune  serum  plus  culture:  plate 

shows  many  thousands  of  colonies. 
Control  8:    0.01  c.c.  of  inactivated  normal  serum  plus  culture: 

many  thousands  of  colonies. 

An  examination  of  this  experiment  shows  that  the  dose  of  culture 
was  satisfactory,  that  the  culture  increased  during  the  three  hours  of 
primary  incubation,  that  the  complement  serum  was  very  slightly 
bactericidal  in  a  dose  lower  than  that  used  in  making  the  test,  and  that 
the  technic  was  fairly  satisfactory,  slight  contamination  being  present 
in  but  one  plate — that  of  the  inactivated  normal  control  serum. 

The  most  striking  result  observed  is  the  absence  of  bactericidal  ac- 
tivity in  the  first  two  plates,  which  contained  the  largest  amount  of 
immune  serum  and  where  one  would  naturally  expect  to  find  complete 
sterility.  The  titer  of  this  serum  was  between  1  : 3200  and  1  : 6400. 
According  to  Neisser  and  Wechsberg,  these  paradoxic  results  are  caused 
by  deviation  or  "  deflection  of  complement,"  as  was  explained  in  a  pre- 
vious chapter.  In  bactericidal  experiments,  according  to  Neisser,  the 
deflection  is  caused  by  an  excess  of  amboceptors  in  the  immune  serum. 
In  a  mixture  of  bacteria,  complements,  and  large  amounts  of  amboceptor 
the  complement  is  bound  not  only  by  the  amboceptors  anchored  to  the 
bacteria,  but  also  in  large  measure  by  "free"  amboceptors  that  are  not 
anchored  to  bacteria.  A  portion  of  the  anchored  amboceptor,  therefore, 
finds  no  complement  at  its  disposal,  and  is,  therefore,  unable  to  exert  any 
bactericidal  action,  which  gives  rise  to  a  relative  lack  of  complement. 

This  phenomenon  resembles  the  action  of  agglutinoids  in  the  agglu- 
tination reaction,  where,  in  the  lowest  dilutions,  agglutination  is  feeble 
or  absent,  but  becomes  manifest  in  the  higher  dilutions. 


TECHNIC    OF   BACTERIOLYTIC    TESTS  377 

In  the  foregoing  experiment  the  immune  serum  used  was  several 
weeks  old;  perfectly  fresh  serums  are  not  so  likely  to  show  this  so-called 
"  deflection  of  complement."  In  many  instances,  and  especially  if  a 
fresh  serum  is  used,  one  cannot  help  thinking  that  agglutinins  may  be 
responsible  for  the  absence  or  diminution  of  bactericidal  activity  in  the 
lower  dilutions.  It  is  certainly  true  that  hemagglutinins  considerably 
inhibit  hemolysis,  and  this  is  especially  the  case  with  serums  of  low  hemo- 
lytic  activity.  With  more  potent  serums  the  agglutinins  are  diluted 
until  activity  ceases  and  hemolysis  is  ready  and  complete.  Reasoning 
from  analogy,  therefore,  the  absence  or  diminution  of  bactericidal  ac- 
tivity may  be  due  to  agglutinins,  and  the  theory  of  "  deflection  of  com- 
plements" may  be  emphasized  a  little  too  strongly. 

According  to  Halm,  normal  human  serums  show  bactericidal  activity 
in  only  about  one-third  of  the  cases,  and  the  titer  is  only  very  exception- 
ally demonstrable  in  dilutions  higher  than  1  : 500.  The  serums  of  ad- 
vanced cases  of  typhoid  fever  or  of  those  but  recently  recovered  are 
bactericidal  in  dilutions  greater  than  1  : 1000,  and  may  reach  1  :  50,000 
or  higher.  Similarly  after  typhoid  immunization  the  patient's  serum 
may  show  a  high  bactericidal  titer.  Weston  has  found  such  serums 
active  in  dilutions  of  1  :  20,000,  higher  dilutions  not  being  used. 

Besides  being  used  in  typhoid,  the  plate  culture  method  has  been 
employed  for  experimental  purposes  in  cholera,  dysentery,  paratyphoid, 
and  other  infections  with  bacilli  of  the  typhoid-colon  group.  With 
these,  however,  the  test  possesses  but  little  diagnostic  significance. 

The  bactericidal  titer  does  not  run  strictly  parallel  with  the  agglu- 
tinins or  complement-fixing  bodies. 

Measuring  the  Bactericidal  Power  of  the  Blood  by  Capillary  Pipet 
Method  (After  Wright).1 — By  this  technic  it  is  sought  to  overcome  the 
fallacies  of  the  "loopful"  method  of  measurement  and  those  due  to 
agglutination  of  the  test  organism. 

The  native  complements  of  the  patient's  own  serum  are  used;  hence 
the  serums  used  in  this  technic  must  be  fresh.  Quantitative  titration  is 
accomplished  by  furnishing  varying  dilutions  of  culture,  with  a  constant 
quantity  of  serum.  A  series  of  volumes  of  serum  is  taken,  and  to  these 
are  added  equal  quantities  of  progressively  increasing  dilutions  of  a 
counted  bacterial  culture.  The  mixtures  are  kept  at  37°  C.,  for  twenty- 
four  hours,  after  which  each  is  introduced  into  nutrient  broth  and  culti- 
vated to  see  whether  a  complete  bactericidal  effect  has  been  exerted. 

1  Wright,  A.  E.:  Technique  of  the  Teat  and  Capillary  Glass  Tube,  1912,  Lon- 
don, Constable  &  Co. 


378  BACTERIOLYSINS 

The  largest  number  of  bacteria  that  a  constant  quantity  of  serum  has  been 
able  to  kill  furnishes  a  measure  as  to  its  bactericidal  power. 

After  a  few  technical  details  have  been  mastered,  this  method  is 
quickly  performed  and  yields  fairly  constant  and  reliable  results.  It  is 
not  adapted  for  the  titration  of  old  immune  serums,  but  is  a  ready  clin- 
ical test,  finding  a  special  field  of  usefulness  in  determining  the  bacteri- 
cidal powers  of  the  blood  after  typhoid  immunization. 

Requisites  for  Carrying  out  the  Test. — 1.  A  specimen  of  the  patient's 
blood  is  collected  aseptically,  a  process  that  may  be  accomplished  by 
thoroughly  cleansing  the  finger  with  alcohol  and  collecting  blood  in  a 
sterile  Wright  capsule,  or  better,  perhaps,  by  means  of  venipuncture, 
when  from  2  to  5  c.c.  may  be  collected  aseptically  in  a  sterile  centrifuge 
tube.  The  control  blood  from  a  healthy  individual  may  be  collected 
in  a  capsule.  The  serums  are  carefully  separated  and  pipeted  into  small 
sterile  test-tubes;  1  c.c.  of  each  is  ample  for  the  test. 

2.  A  twenty-four-hour-old  broth  culture  of  the  test  organism  (a 
young  culture  is  required,  because  such  a  culture  contains  a  few  dead 
microorganisms  and  the  absorption  of  bactericidal  elements  by  dead 
organisms  is  thus  avoided). 

3.  About  two  dozen  "  looped  pipets,"  made  according  to  the  direc- 
tions given  in  Chapter  I. 

4.  Sterile  neutral  broth  for  making  dilutions  and  cultivations.    This 
is  prepared  and  sterilized  in  the  usual  manner,  from  5  to  10  c.c.  being 
placed  in  test-tubes.     When  working  with  the  typhoid  bacillus  a  special 
broth  containing  1  per  cent,  of  mannite  and  sufficient  litmus  to  color 
it  a  deep  blue  (Smallman)  should  be  on  hand. 

5.  Two  dozen  small  test-tubes,  plugged  and  sterilized,  for  making 
dilutions  of  the  culture. 

Preparation  and  Enumeration  of  the  Bacterial  Culture. — As  the  prin- 
ciple of  the  test  depends  upon  measuring  the  bactericidal  activity  of  the 
blood  according  to  the  number  of  organisms  that  are  killed,  it  is  neces- 
sary to  prepare  somewhat  high  dilutions  and  count  the  number  of 
organisms  in  a  unit  volume  in  each  dilution. 

For  this  purpose  place  10  sterile  test-tubes  in  a  rack,  and  arrange  10 
sterile  Petri  dishes  on  the  table  to  correspond  to  these.  To  the  first 
tube  add  4.9  c.c.  of  plain  sterile  broth;  to  the  second,  fourth,  sixth, 
eighth,  and  tenth  tubes  add  1  c.c.,  to  the  third,  fifth,  seventh,  and  ninth 
tubes,  add  4  c.c. 

With  a  sterile  graduated  1  c.c.  pipet  add  0.1  c.c.  culture  to  the  first 
tube  and  then  discard  the  pipet  by  placing  it  in  a  cylinder  of  disinfecting 


! 


II 


FIG.  104.— BACTERICIDAL  TEST  (LOOPED  PIPET  METHOD  OF  WRIGHT). 
The  pipet  shown  on  the  extreme  left  contains  the  mannite-litmus  broth  and  an 
equal  volume  of  patient's  serum  and  emulsion  of  typhoid  bacilli;  the  second  pipet 
shows  the  serum  and  bacillary  emulsion  mixed;  the  middle  pipet  shows  the  serum- 
bacillary  mixture  cultured  in  the  broth;  the  fourth  pipet  shows  acid  production  in 
the  broth  by  living  bacilli  which  escaped  destruction  after  twenty-four  hours'  incuba- 
tion; the  fifth  pipet  (extreme  right)  is  the  serum  control.  The  broth,  being  clear 
and  unchanged,  shows  that  the  serum  was  sterile. 


TECHNIC    OF   BACTERIOLYTTC    TESTS  379 

solution  or  hot  water.  With  a  second  sterile  pipet  mix  the  contents  of 
tube  No.  1  by  carefully  sucking  it  in  and  forcing  it  out  of  the  pipet  sev- 
eral times,  and  place  0.1  c.c.  in  Petri  dish  No.  1.  Then,  with  the  same 
pipet,  transfer  1  c.c.  to  the  second  tube,  mix  well,  and  place  0.1  c.c.  in 
Petri  dish  No.  2;  next  transfer  1  c.c.  to  the  third  tube,  mix,  and  place 
0.1  c.c.  in  the  third  Petri  dish.  Continue  in  this  way  until  the  last  tube 
is  reached,  when  1  c.c.  is  discarded  into  a  germicidal  solution  and  0.1 
c.c.  placed  in  the  tenth  Petri  dish.  To  each  Petri  dish  now  add  from  8 
to  10  c.c.  of  neutral  agar  at  41°  C.,  and  mix  thoroughly.  After  the 
plates  have  hardened  they  are  turned  over  in  order  that  the  water  of 
condensation  may  collect  on  the  cover.  They  are  then  incubated  for 
twenty-four  hours,  and  the  colonies  carefully  counted. 

We  now  have  the  following  dilutions  of  culture :  1  : 50,  1  : 100, 
1 :  500,  1  :  1000,  1  :  5000,  1  :  10,000,  1  :  50,000,  1  :  100,000,  1  :  500,000, 
and  1  :  1,000,000.  Since  but  0.1  c.c.  of  these  dilutions  were  plated,  the 
total  number  of  colonies  in  each  plate  must  be  multiplied  by  10  in  order 
to  obtain  the  approximate  number  per  cubic  centimeter  of  the  various 
dilutions. 

To  secure  fairly  uniform  results,  the  various  dilutions  must  be 
thoroughly  mixed  and  the  pipeting  be  accurately  performed.  We  have 
found  that  this  method  usually  gives  better  results  than  are  obtained 
by  plating  but  one  or  two  of  the  higher  dilutions,  which  are  used  as  a 
basis  for  calculating  the  number  of  bacteria  in  the  other  dilutions. 

Filling  the  Pipets. — With  a  wax  pencil  make  a  mark  upon  the  capillary 
stem  of  a  sterile  looped  pipet  at  a  point  2  cm.  from  the  lower  end,  and 
fit  a  rubber  teat  to  the  barrel.  The  point  of  the  capillary  stem  is  now 
broken  off  between  the  finger  and  thumb,  the  lower  portion  is  sterilized 
in  the  flame,  and  the  air  is  expelled  from  the  teat. 

Mannite  broth  is  then  aspirated  into  the  pipet  until  the  bulb  is  about 
two-thirds  full  and  the  capillary  portion  contains  an  air  column  rising 
to  at  least  5  cm.  from  the  end  (Fig.  104). 

The  end  of  the  capillary  stem  is  now  inserted  into  the  tube  contain- 
ing the  patient's  serum,  and  the  serum  is  allowed  to  flow  in  until  it 
reaches  the  pencil  mark. 

The  orifice  of  the  pipet  is  now  raised  above  the  surface  of  the  serum, 
and  a  small  bubble  of  air  is  admitted  into  the  tube,  to  serve  as  an  index 
for  the  measurement.  The  end  of  the  capillary  stem  is  now  carried 
into  the  tube  containing  the  highest  dilution  of  the  organism,  and  the 
culture  is  allowed  to  flow  in  until  the  bubble  of  air  has  been  carried  just 
past  the  pencil  mark. 


380  BACTERIOLYSINS 

The  serum  and  culture  must  now  be  mixed,  being  careful  not  to  con- 
taminate the  broth.  This  is  effected  by  blowing  these  two  volumes  out 
into  a  sterile  mixing  tube  and  drawing  up  and  blowing  out  the  fluid 
several  times  in  succession.  By  starting  with  the  highest  dilution,  the 
same  mixing  tube  may  be  used  throughout  the  series.  With  an  air- 
bubble  of  at  least  5  cm.  between  serum  and  broth,  this  can  be  quite  easily 
achieved  without  driving  the  sterile  broth  down  from  the  bulb  of  the 
pipet  into  the  lower  part  of  the  capillary  stem  and  contaminating  it  there. 

The  column  of  mixed  serum  and  culture  is  now  drawn  up  into  the 
middle  region  of  the  capillary  stem  and  the  lower  end  of  the  tube  is 
sealed.  The  teat  is  then  removed,  and  the  dilution  that  has  been  used 
is  written  on  the  barrel  of  the  pipet. 

The  series  of  pipets  are  now  filled  with  the  nine  measuring  dilutions 
of  the  culture. 

Controls. — The  serum  from  a  healthy  individual,  or,  better  still,  the 
pooled  serums  of  several  persons,  may  be  used  in  precisely  the  same  way, 
with  at  least  the  second,  fourth,  sixth,  and  eighth  dilutions  of  culture. 

Several  culture  controls  are  advisable.  The  pipets  are  filled  with 
mannite  broth  in  the  usual  manner,  and  then  with  one  volume  of  at  least 
four  different  dilutions,  usually  1  :  50,  1  :  5000,  1  : 100,000,  and  1  :  1,000,- 
000.  The  tubes  are  sealed,  labeled,  and  incubated  together  with  those 
concerned  in  the  test  proper. 

One  pipet  is  to  contain  the  usual  quantity  of  broth  and  one  volume 
of  the  patient's  serum.  This  is  a  control  on  the  sterility  of  the  patient's 
serum.  A  similar  preparation  is  made  with  the  control  serum  to  serve 
as  a  control  on  its  sterility. 

When  the  whole  series  of  tubes  have  been  filled,  these  are  placed 
upright  in  a  large  test-tube  or  cylinder  labeled  with  the  date  and  the 
source  of  the  serum,  and  incubated  at  37°  C.  for  from  eighteen  to  twenty- 
four  hours. 

Test  for  the  Germicidal  Activity  of  the  Serum. — The  serum  and  culture 
in  the  capillary  portion  of  the  tube  must  now  be  mixed  with  the  broth  in 
the  barrel.  This  is  accomplished  by  taking  each  pipet  in  hand  singly, 
and  heating  the  lower  portion  of  the  capillary  stem  in  a  " peep-flame" 
and  drawing  out  into  a  small  thread  with  a  pair  of  forceps. 

A  collapsed  teat  is  now  fitted  over  the  barrel,  and  the  negative  pres- 
sure carefully  regulated  by  keeping  the  finger  and  thumb  in  position  on 
the  teat,  and  the  finely  drawn  out  end  of  the  capillary  stem  gently 
snapped  across.  The  column  of  serum  and  culture  will  then  be  carried 
up  into  the  bulb  of  the  pipet.  The  end  of  the  tube  is  now  sealed. 


BACTERIOLYTIC   SERUMS  IN  THE   TREATMENT  OF  DISEASE       381 

When  the  whole  series  of  pipets  has  been  dealt  with  in  similar  fashion 
they  are  returned  to  the  incubator  for  another  twenty-four  hours. 

Reading  the  Result. — The  continued  sterility  of  the  broth  may  be  de- 
termined from  direct  naked-eye  inspection. 

When  a  complete  bactericidal  effect  has  been  secured,  the  broth  will 
have  undergone  no  color  change,  but  will  still  be  clear. 

When  a  growth  of  the  typhoid  bacillus  has  occurred,  a  perceptible 
cloudiness  will  be  apparent  in  the  broth,  and  the  color  will  have  changed 
from  blue  to  reddish-blue,  indicating  that  acid  has  been  formed  from  the 
mannite. 

Turbidity  of  the  broth  without  a  change  of  color  would  indicate  the 
admission  of  contaminating  microbes  that  were  unable  to  split  mannite 
with  the  formation  of  acid. 

The  controls  are  first  examined  and  recorded.  All  the  culture  con- 
trols should  show  a  growth  and  change  of  color.  The  serum  controls 
should  be  sterile;  the  normal  serum  controls  may  or  may  not  be  sterile, 
depending  upon  the  amount  of  natural  bactericidal  amboceptors  which 
it  contains  for  the  test  organism. 

The  pipets  containing  the  patient's  serum  are  now  examined,  and  a 
simple  numeric  expression  for  the  result  is  obtained  by  referring  to  the 
result  of  the  enumeration  of  the  various  dilutions,  as  determined  by  the 
counting  of  the  plates.  In  this  manner  the  number  of  bacteria  contained 
in  1  c.c.  of  the  lowest  dilution  of  the  bacterial  culture  that  has  been  com- 
pletely sterilized  is  calculated.  This  will  give  the  number  of  micro- 
organisms which  1  c.c.  of  serum  would  be  capable  of  killing. 

Although  this  test  affords  a  convenient  basis  for  the  comparison  of 
serums,  it  must  be  understood  that  the  expression  is  entirely  arbitrary, 
and  will  vary  according  to  the  culture  employed;  this  is  true  of  any 
technic  for  determining  the  bactericidal  titer  of  a  serum. 


BACTERIOLYTIC  SERUMS  IN  THE  TREATMENT  OF  DISEASE 
While  bacteriolysis  is  readily  demonstrated  with  some  bacteria  both 
in  vivo  and  in  vitro,  nevertheless,  when  such  serums  are  used  thera- 
peutically,  beneficial  results  are  not  dependent  solely  upon  lysis  of  the 
infecting  bacterium,  but  are  usually  the  result  of  a  combination  of  lysis 
with  increased  phagocytosis,  due  to  the  simultaneous  presence  of  bac- 
teriotropins  (immune  opsonins).  When  one  recalls  the  close  similarity 
in  general  properties  that  exists  between  bacteriolysins  and  bacterio- 
tropins,  the  difference  between  extracellular  and  intracellular  lysis  is 


382  BACTERIOLYSINS 

relatively  slight,  and  tends  to  strengthen  Metchnikoff's  views  regarding 
the  close  relationship  of  the  bacteriolysins  to  leukocytic  products  and 
phagocytosis.  While  extracellular  lysis  can  occur  in  the  absence  of 
cells,  especially  in  vitro,  yet  in  the  administration  of  antibacterial  serums 
the  activities  of  the  leukocytes  are  much  in  evidence,  extracellular  lysis 
or  bacteriolysis  proper  being  rather  subsidiary  to  intracellular  lysis  or 
phagocytosis. 

The  preparation  and  methods  of  standardization  of  antibacterial 
serums  are  given  in  the  chapter  on  Passive  Immunization.  Most  effort 
in  this  direction  has  been  expended  on  the  preparation  of  antiserums  for 
the  treatment  of  meningococcic,  streptococcic,  pneumococcic,  and 
gonococcic  infections,  and  the  greatest  success  has  been  attained  with 
the  various  antimeningococcic  serums. 


CHAPTER  XX 
HEMOLYSINS 

Hemolysis  is  the  term  applied  to  the  solution  or  lysis  of  red  blood- 
corpuscles.  Strictly  speaking,  it  would  include  the  disintegration  and 
solution  of  the  stroma,  although  in  practice  the  term  is  applied  to  any 
process  in  which  the  cells  are  so  injured  as  to  liberate  hemoglobin  into 
the  surrounding  fluids,  with  or  without  solution  of  the  stroma. 

Hemolysis  may  be  caused  by  various  physical,  chemical,  and  specific 
agencies.  The  prolonged  agitation  of  blood  with  glass  beads,  for  in- 
stance, may  result  in  the  mechanical  rupture  of  erythrocytes.  The 
addition  of  blood  to  hypotonic  solutions  of  sodium  chlorid  or  to  plain 
water,  results  in  ready  and  complete  hemolysis,  the  fluid  being  trans- 
formed from  an  opaque  red  suspension  of  erythrocytes  to  a  clear,  trans- 
parent red  fluid.  Various  chemicals,  such  as  acids  and  alkalis,  may  also 
produce  hemolysis.  As  previously  stated,  a  few  bacterial  toxins,  such 
as  tetanolysin  and  staphylolysin,  are  known  to  be  hemolytic;  the  same 
is  true  of  certain  vegetable  toxins,  such  as  abrin  and  ricin,  and  of  some 
animal  toxins,  such  as  cobra,  toad,  and  scorpion  venoms. 

Just  as  bacteria  may  be  killed  and  possibly  broken  up  by  specific 
bacteriolytic  amboceptors  and  complements,  so,  in  like  manner,  hemol- 
ysis may  be  caused  by  specific  hemolytic  antibodies  known  as  hemolysins. 
Working  in  unison  with  complements,  the  mechanism  of  both  bacterio- 
lysis and  serum  hemolysis  is  probably  identical.  The  simplicity  of 
hemolytic  experiments,  the  rapidity  with  which  they  may  be  performed 
and  terminated,  and  the  ease  with  which  hemolysis  may  be  observed 
by  the  naked  eye  have  rendered  the  specific  serum  hemolysins  particu- 
larly useful  in  the  study  of  amboceptors  and  of  complements,  and  of 
cytolytic  phenomena  in  general.  In  fact,  bacteriolysis  was  not  thor- 
oughly understood  until  Bordet  discovered  the  hemolysins,  and  demon- 
strated the  analogy  that  exists  between  bacteriolysis  and  hemolysis,  a 
discovery  that  led  to  a  vast  amount  of  research  work  and  controversy, 
to  many  important  discoveries,  and  to  the  final  evolvement  of  diagnostic 
reactions  of  great  value. 

Historic. — For  many  years  physiologists  were  aware  that  the  bloods 
of  various  animals  transfused  into  man  or  animals  of  a  different  species 

383 


384  HEMOLYSINS 

were  more  or  less  directly  injurious,  and  incapable  of  replacing  human 
blood.  In  1875  Landois  demonstrated  experimentally  that  while  trans- 
fusion of  blood  from  one  animal  to  another  of  a  different  species  may 
prove  injurious  and  even  fatal,  transfusion  to  an  animal  of  the  same  or  of 
very  closely  related  species  produced  no  ill  effects.  The  explanations 
offered  were  inadequate,  until  later  researches  on  the  hemolysins  showed 
that  the  normal  blood-serum  of  one  animal  may  contain  hemolysins  for 
the  erythrocytes  of  other  animals,  and,  consequently,  upon  transfusing 
this  blood  to  another  animal  the  hemolysin  acting  with  the  complement 
present  produced  hemolysis  in  vitro,  thereby  explaining  in  part  the  tox- 
icity  of  the  alien  blood. 

In  1898  Belfanti  and  Carbone  made  the  observation  that  the  serum 
of  a  horse^receiving  several  injections  of  rabbit  bloocpwas  toxic  for  rab- 
bits, whereas  normal  horse  serum  was  without  injurious  effects. 

At  about  the  same  time  Bordet  published  his  epoch-making  discov- 
eries. He  observed  that  while  normal  guinea-pig  serum  had  little  or 
no  hemolytic  action  on  rabbit  erythrocytes,  the  serum  ofji  pig  that  had 
received  a  few  intraperitoneal  injections  of  rabbit  blood  was  able  quickly 
and_comrjletely  to  hemolyze  rabbit  bloody  ust  as  an  animal  may  acquire, 
through  immunization  with  cholera,  the  property  of  dissolving  cholera 
vibrios.  He  demonstrated  further  that  this  acquired  hemolytic  activity 
.was  highly  specific,  for  when  animal  A  was  immunized  with  the  corpuscles 
of lanimal  B  the  serum  of  A  acquired  the  power  of  hemolyzing  only  the 
erythrocytes  of  B,  and  possibly  of  other  animals  closely  related  zoologic- 
ally. It  was  found  also  that  the  hemolytic  activity  of  an  immune  serum 
was  lost  by  age  or  could  be  removed  by  heating;  that  in  either  case  the 
serum,  could  be  reactivated  by  the  addition  of  a  little  normal  serum  or. 
jperitoneal  exudate — phenomena  closely  resembling  that  observed  in 
iiacteriolysis,  and_due  to  the  action  of  a  thermolabile  body  or  alexin  and 
a  second  and  specific  thermostabile  antibody  named  by  Bordet  the 
"substance  sensibilisatrice." 

These  observations  were  soon  confirmed  by  Landsteiner  and  von 
Dungern,  and  were  followed  by  very  extensive  studies  by  Ehrlich  and 
Morgenroth,  who  likewise  confirmed  Bordet 's  experiments,  but  offered  a 
different  explanation  for  the  mechanism  of  the  phenomenon,  according 
to  the  side-chain  theory,  and  renamed  the  alexin  and  sensitizing  sub- 
stance concerned  in  the  process  "complement"  and  "hemolytic  ambo- 
^ceptor"  or  "immune  body,"  respectively. 

Definition. — Hemolysins  [Gr.,  alpa  =  blood  +  \veiv  =  to  dissolve] 
are  antibodies  in  a  serum  that,  when  acting  with  complement,  have  the 


NATURE    OF   HEMOLYSINS  385 

power  of  "lysing"  or  breaking  up  red  blood-corpuscles,  or  so  altering  their 
envelop  as  to  allow  the  hemoglobin  to  escape. 

Nomenclature. — Normal  hemolysins  are  those  found  in  normal  se- 
rums; specific  or  immune  hemolysins  are  those  produced  as  the  result  of 
the  injection  of  blood-corpuscles  from  an  animal  of  a  different  species.  ,  / 
Heterolysins  are  the  hemolysins  formed  by  immunization  with  corpuscles  (/ 
of  a  different  species  (the  immune  hemolysins).  Isolysins  are  hemoly- 
sins for  the  corpuscles  of  animals  of  the  same  species.  Autolysins  are 
hemolysins  that  act  upon  the  corpuscles  of  the  same  anima.1  and  are  quite 
rare.  It  should  be  remarked  that  isolysin  and  autolysin  are  not  strictly 
synonymous  terms,  as  the  former  does  not  act  upon  the  corpuscles  of  the 
animal  producing  the  hemolysin,  but  may  hemolyze  the  corpuscles  of 
other  animals  of  the  same  species.  For  example,  Ehrlich  has  shown  that 
the  serum  of  a  goat  that  had  received  several  injections  of  blood  from 
other  goats,  although  actively  hemolytic  for  the  corpuscles  of  goats  1,  2, 
4,  5,  6,  9,  and  less  so  for  goats  3  and  8,  was  not  able  to  hemolyze  those  of 
goat  7  or  of  itself  at  all.  This  immunity  of  the  corpuscles  of  an  animal 
to  its  own  isolysin  was  subsequently  shown  to  be  due  to  a  complete 
absence  of  suitable  receptors  in  its  corpuscles.  Therefore  in  cases  in 
which  a  large  internal  hemorrhage  occurs  and  the  blood  is  absorbed  an 
autohemolysin  is  not  produced,  or  produced  only  in  small  amounts,  and 
likely  to  be  followed  by  the  formation  of  an  anti-autolysin  which 
regulates  the  process  of  blood  destruction  within  physiologic  limits. 
Although  little  is  known  concerning  the  processes  of  normal  blood  destruc- 
tion, as  in  the  disposal  of  old  erythrocytes,  it  is  possible  that  an  auto- 
hemolysin is  being  produced,  and  that  its  activity  is  held  within  normal 
limits  by  an  anti-autolysin.  A  disturbance  of  this  physiologic  equilib- 
rium may  then  be  the  basis  of  certain  types  of  primary  anemia  character- 
ized by  excessive  blood  destruction. 

Nature  of  Hemolysins. — According  to  the  side-chain  theory,  he- 
molysins are  amboceptors  or  antibodies  of  the  third  order,  requiring  the 
action  of  a  complement  before  hemolysis  can  be  produced  (Fig.  105). 
Bordet  showed  that  two  substances  were  concerned  in  the  phenomenon 
of  serum  hemolysis,  although  his  views  on  the  mechanism  of  the  process 
differ  from  those  advanced  by  Ehrlich  and  Morgenroth. 

Ehrlich  argued  that  the  hemolytic  amboceptor  or  hemolysin  must 
be  an  antibody  to  the  receptors  of  the  red  blood-corpuscles  used  in  the 
process  of  immunization,  and  if  this  is  true,  it  ought  to  unite  with  the 
corpuscles.  Taking  the  serum  of  a  goat  that  had  been  injected  with  and 
was  hemolytic  for  the  erythrocytes  of  a  sheep,  he  destroyed  the  comple- 
25 


386  HEMOLYSINS 

ment  by  heating  the  serum  to  56°  C.  To  this  he  added  some  sheep's 
corpuscles,  and  allowed  the  mixture  to  stand  for  a  short  time  at  room 
temperature,  after  which  it  was  centrifuged  and  the  supernatant  fluid 
pipeted  off  in  another  test-tube.  No  hemolysis  had  occurred,  and  the 
corpuscles  were  to  all  appearances  unaltered,  but  it  was  now  found  that 
if  a  small  amount  n£jTnrjmfl,1  gn^t^ggTrinv,  as  complement,  was  added  to 
the  corpuscles  and  the  mixture  placed  in  the  incubator,  hemolysis  oc- 
curred. By  adding  sheejp^s_corpuscles_and  normal  goat  serum  (comple- 
ment) to  the  supernatant  fluid  that  had  been  removed  to  a  separate  test- 
tube,  hemolysis  did  not  occur.  This  experiment  indicated,  therefore, 
that  the  red  blood-corpuscles  had  combined  with  all  the  antibody. 
That  the  action  was  specific  was  shown  by  the  fact  that  the  corpuscles 
of  other  animals,  such  as  rabbits  or  goats,  for  example,  exerted  no  com- 
bining power  when  used  instead  of  the  sheep's  cells.  The  union  between 

cell  and   antibody  was    considered  as 
being  in  the  nature  of  a  chemical  com- 
bination and  quite   firm,   as   repeated 
.     washing  of  the  cells  with  normal  salt 

0  solution  did  not  break  it  up. 
FIG.  105.  —THEORETIC  STRUCTURE 

OF  A  HEMOLYTIC  AMBOCEPTOR  Having   shown   that   the    antibody 

(HEMOLYSIN).  had  &  cpmbining  amnity  for  the   cells, 

A,  Amboceptor;    C,  comple-  *- 

ment;  r,  receptor  of  the  corpuscle;  it  was  important  to  solve  the  question 


of  the  relati™  of  alexin  OT  complement 
group  of  the  amboceptor.  to  the  process.  In  other  words,  does 

this  substance  unite  directly  with  the 
cell  or  does  it  unite  with  the  antibody  and  thus  indirectly  with  the  cell? 

Ehrlich  and  Morgenroth  studied  this  by  means  of  a  similar  experi- 
ment. Sheep's  corpuscles  were  mixed  with  normal  goat  serum  (com- 
plement), and  after  a  time  the  mixture  was  centrifuged  and  the  two  por- 
tions tested  separately.  To  the  corpuscles  heated  immune  serum  was 
added,  but  hemolysis  did  not  result.  To  the  supernatant  fluid  cor- 
puscles and  heated  immune  serum  were  added  and  hemolysis  occurred. 
This  indicated  that  the  alexin  or  complement  did  not  unite  with  the 
corpuscles,  as  did  the  antibody  in  the  first  experiment,  but  remained 
free  in  the  supernatant  fluid. 

By  mixing  corpuscles,  immune  serum,  and  complement  and  keeping 
the  mixture  at  0°-3°  C.  for  several  hours,  hemolysis  did  not  take  place. 
By  centrifuging  the  mixture  and  separating  the  supernatant  fluid  from 
the  corpuscles,  a  similar  test  showed  that  the  red  cells  had  combined 
with  all  the  antibody,  but  had  left  the  alexin  practically  undisturbed. 


NATURE    OF   HEMOLYSINS  387 

At  higher  temperatures  these  relations  were  more  difficult  to  demon- 
strate, as  hemolysis  occurs  rapidly,  but  by  leaving  the  cells  and  serum  in 
contact  for  short  periods  of  time,  centrifuging  rapidly,  and  testing  the 
corpuscles  and  supernatant  fluid  in  the  same  manner,  similar  relations 
were  found  to  exist. 

These  experiments  led  Ehrlich  to  formulate  the  theory  that  comple- 
ment will  unite  only  with  the  antibody  and  not  with  the  red  corpuscles, 
but  that  it  acts  upon  the  corpuscles  when  united  indirectly  by  means  of 
the  antibody.  As  will  be  pointed  out  further  on,  this  view  is  in  direct 
opposition  to  that  of  Bordet,  who  does  not  accept  this  interpretation, 
but  believes  that  the  complement  acts  directly  upon  the  corpuscles. 

Ehrlich,  therefore,  conceived  the  antibody  as  being  in  the  nature  of 
an  amboceptor  or  of  an  interbody  between  an  antigen  and  complement, 
with  two  combining  arms — one  the  cytophile  haptophore  for  union  with 
the  cell,  and  the  second,  the  complementophile  haptophore  for  union  with 
complement.  The  amboceptor  is  unable  in  itself  to  injure  the  cell,  but 
preserves  its  importance  in  being  the  only  and  specific  means  by  which 
the  ferment  or  complement  can  attack  the  cell  and  cause  its  destruction. 

The  process  of  specific  serum  hemolysis  is  therefore  supposed  to  be 
as  follows:  In  fresh  immune  serum  containing  both  amboceptors  and 
complement,  or  in  a  mixture  of  old  or  heated  immune  serum  and  fresh 
normal  serum  (i.  e.,  of  amboceptor  and  complement),  the  two  substances 
occur  independently  of  each  other.  When  the  corpuscles  corresponding 
to  the  amboceptor  are  added,  the  amboceptor  unites  with  these  and  the 
complement  unites  with  the  amboceptor,  the  amboceptor,  therefore, 
standing  midway  between  corpuscles  and  complement.  When  these 
unions  have  taken  place,  hemolysis  will  result.  The  amboceptor  has  a 
greater  affinity  for  the  corpuscles  than  the  complement  has  for  the  am- 
boceptor, and  will  unite  with  the  cells  at  a  low  temperature,  whereas  the 
complement  unites  with  the  amboceptor  only  very  slowly  at  low  tem- 
peratures. Body  temperature  favors  a  quicker  union  of  both,  and 
especially  that  of  complement  with  amboceptor.  Hemolysis,  therefore, 
may  occur  at  low  temperatures,  but  is  hastened  by  higher  temperatures, 
and  occurs  best  at  37°  C. 

As  stated  in  a  previous  chapter,  Ehrlich  believes  that  a  great  many 
complements  exist  in  normal  serums,  which  view  is  in  direct  opposition 
to  the  "unitarian  theory"  of  Bordet,  which  holds  that  the  one  alexin  or 
complement  will  act  with  any  sensitizer  or  amboceptor.  Of  the  large 
number  of  complements,  each  is  especially  adapted  for  the  solution  of 
one  or  more  varieties  of  cells,  which  it  can  dissolve  in  conjunction  with  a 


388  HEMOLYSINS 

suitable  amboceptor.  This  is  known  as  the  main  or  dominant  comple- 
ment; other  complements  that  may  aid  in  the  process  are  termed  non- 
dominant.  In  general,  it  is  held  that  those  complements  that  are 
especially  active  in  hemolysis  are  but  slightly  active  in  bacteriolysis,  and 
vice  versa.  Although  the  amboceptor  is  depicted  as  having  but  one 
haptophore  arm  for  the  dominant  complement,  it  is  really  a  polyceptor, 
and  is  so  constituted  that  it  combines  with  the  cell  to  be  dissolved,  on  the 
one  hand,  and  with  a  number  of  complements,  on  the  other. 

Bordet  has  never  accepted  these  views.  He  holds  that  the  antibody 
is  not  an  amboceptor  for  uniting  cell  and  complement,  but  that  it  sensi- 
tizes the  cell  and  renders  it  susceptible  to  the  direct  lytic  action  of  the 
alexin  or  complement.  According  to  his  views,  both  antibody  and 
complement  may  unite  directly  with  the  cell,  and  he  has  borne  out  this 
belief  by  making  experiments  almost  exactly  similar  to  those  made  by 
Ehrlich. 

In  accepting  Ehrlich's  view,  it  is  a  question  of  considerable  practical 
importance  whether  the  complement  may  unite  directly  with  free  am- 
boceptor. Ehrlich  maintains  that  the  two  may  enter  into  a  loose  and 
easily  dissociated  chemical  combination,  which  is  hastened  by  heat  and 
retarded  by  cold.  The  union  of  hemolytic  amboceptor  in  cobra  venom 
with  the  lecithin  of  corpuscles  (Kyes'  cobra  lecithid),  which  acts  as  com- 
plement, while  it  tends  to  strengthen  this  view  can  hardly  be  accepted 
as  direct  proof,  as  lecithin  differs  markedly  from  the  ordinary  comple- 
ments found  free  in  serum.  Likewise  the  theory  of  Neisser  and  Wechs- 
berg  regarding  complement  deviation,  whereby  it  appears  that  an  excess 
of  amboceptor  may  combine  directly  with  complement  and  in  this 
manner  rob  those  amboceptors  that  are  attached  to  cells  of  the  comple- 
ment necessary  to  produce  lysis,  is  quite  complicated,  and  is  not  uni- 
versally accepted,  the  evidence  of  direct  union  of  complement  and  am- 
boceptor having  not  been  proved  beyond  the  peradventure  of  a  doubt. 
It  follows,  then,  as  Emery  has  stated,  thai/ we  must  either  assume  that 
the  complementophile  haptophore  of  an  amboceptor  united  with  its 
antigen  has  an  increased  affinity  for  complement  over  and  above  that  of 
free  amboceptors,  or  we  must  agree  with  Bordet  that  cell  and  comple- 
ment unite  directly  after  the  former  has  been  sensitized  by  the  action  of 
the  antibody.  This  latter  view  of  the  separate  union  of  cell  with  anti- 
body and  complement  is  supported  by  the  observations  of  Muir,  who 
found,  (upon  saturating  red  blood-corpuscles  with  antibody  and  then  with 
complement,  that  some  of  the  former  but  none  of  the  latter  may  be  dis- 
sociated from  the  combination  and  become  free  in  the  fluid.  However, 


NATURE    OF   HEMOLYSINS         .  389 

the  dissociated  amboceptors  found  free  of  complement  may  be  those  that 
did  not  have  time  to  unite  with  complement,  and  there  does  not  appear 
to  be  any  direct  and  positive  experimental  evidence  to  permit  one  to 
decide  between  Ehrlich's  and  Bordet's  views. 

While  Ehrlich  believes  that  the  union  between  cell  and  amboceptor 
is  a  chemical  one  and  follows  ordinary  chemical  laws,  obeying  the  law 
of  multiple  proportions,  Bordet  holds  that  the  antibody  acts  as  a  mor- 
dant and  sensitizes  the  cell,  comparing  the  process  to  the  staining  of 
filter-paper  when  immersed  in  a  dye,  or  to  the  use  of  mordants  prepara- 
tory to  the  staining  of  flagella  of  certain  bacteria.  For  example,  0.4 
c.c.  of  a  hemolytic  serum,  if  added  at  once,  was  found  to  dissolve  0.5 
c.c.  of  corpuscles.  If  0.2  c.c.  of  corpuscles  were  first  added,  and  amounts 
of  0.1  c.c.  were  added  subsequently,  no  lysis  took  place  after  that  of  the 
first  portion  added.  Bordet  cited  this  as  an  example  of  a  physical  pro- 
cess of  the  nature  of  absorption,  just  as  filter-paper  when  added  at  once 
to  a  dye  will  be  stained  a  uniform  color,  whereas  if  it  be  added  a  little  at 
a  time,  the  first  pieces  inserted  will  be  stained  deeply,  the  subsequent 
ones  less  and  less  so,  until  the  dye  is  completely  absorbed.  Recent  re- 
searches on  the  colloidal  theory  of  antibodies  would  indicate  that  the 
hemolysins  are  governed  by  very  complex  chemicophysical  laws,  not  as 
yet  fully  understood,  which  regulate  the  action  of  colloids  on  one  another 
and  are  probably  intimately  concerned  in  the  processes  under  discussion. 

As  was  stated  in  a  previous  chapter,  Metchnikoff  maintains  that  both 
substances  concerned  in  hemolysis  are  ferments,  and  that  both  are 
adapted  for  intracellular  digestion.  He  regards  complement  or  his  cy- 
tase  as  a  digestive  ferment  derived  from  leukocytes,  and  believes  that  it 
is  set  free  only  when  leukocytes  are  dissolved  (phagolysis),  either  as 
the  result  of  the  injection  of  a  foreign  substance  or  during  the  process  of 
coagulation.  Amboceptor  or  his  "fixateur"  is  likened  to  enterokinase, 
and  like  it  acts  as  an  accessory  ferment  that  unites  the  more  potent  fer- 
ment (cytase)  to  the  particle  to  be  digested.  He  also  regards  it  as  being 
derived  from  leukocytes,  and  considers  that  the  amount  formed  depends 
upon  the  degree  of  phagocytosis  that  occurs  during  the  absorption  of  the 
antigen.  In  the  conception  of  immunity  as  being  fundamentally  a  pro- 
cess of  nutrition,  and  in  the  belief  of  the  existence  of  more  than  one  com- 
plement, the  similarity  between  the  views  of  Ehrlich  and  those  of  Metch- 
nikoff is  indeed  striking. 

Analogy  between  Bacteriolysis  and  Hemolysis. — Studies  in  hemol- 
ysis aided  greatly  in  a  correct  understanding  of  the  mechanism  of  bac- 
teriolysis. It  became  apparent  that  two  substances  were  concerned  in 


390  HEMOLYSINS 

bacteriolysis — one,  the  thermostabile  amboceptor  present  in  the  immune 
serum,  and  the  second,  thermolabile  alexin  or  complement,  furnished 
by  the  peritoneal  exudate  in  the  Pfeiffer  test,  or  by  any  fresh  normal 
serum  in  the  test-tube  bacteriolytic  reaction.  The  discovery  and  study 
of  the  specific  serum  hemolysins  aided  greatly  in  a  better  understanding 
of  bacteriolysis  and  cytolysis  in  general. 

Specificity  of  Hemolysins.— The  hemolysins  are  highly  specific  anti- 
bodies, and  although  partial  or  group  hemolysins  may  be  formed  and 
act  upon  the  corpuscles  of  closely  related  species,  as  antihuman  hemolysin 
on  the  corpuscles  of  the  higher  apes,  yet  the  main  hemolysin  may  be  so 
potent  that  high  dilution  of  the  immune  serum  practically  rules  out  the 
activities  of  group  hemolysins,  the  hemolytic  amboceptor  proving  highly 
specific  for  the  alien  corpuscles  responsible  for  its  production. 


NORMAL  HEMOLYSINS 

Just  as  small  amounts  of  antitoxins,  agglutinins,  and  opsonins  may 
be  found  in  normal  serums,  so,  also,  normal  hemolysins  may  be  present. 
Their  most  important  practical  significance  is  concerned  with  comple- 
ment-fixation reactions,  where  a  close  and  intimate  quantitative  rela- 
tion exists  between  the  complement  and  hemolytic  amboceptor  used. 
As  an  excess  of  hemolytic  amboceptor  may  produce  hemolysis  with  a 
decreased  amount  of  complement,  in  a  given  test  for  free  complement,  as 
in  Wassermann's  syphilis  reaction,  the  patient's  serum  may  contain  so 
much  natural  antisheep  amboceptor  as  to  make  up  for  slight  binding  of 
complement  and  give  undue  hemolysis  or  even  a  false  negative  result. 

Hemolysis  of  alien  corpuscles  by  a  normal  serum  is  found  to  depend 
upon  the  same  mechanism  of  amboceptor  and  complement  as  in  the  ar- 
tificial immune  serums.  The  amboceptors  are  easily  removed  by  adding 
the  corresponding  corpuscles  to  the  cold  serum  and  centrif  uging  the  mix- 
ture after  allowing  it  to  stand  at  0°-5°  C.  for  an  hour  or  two.  The  su- 
pernatant fluid  will  now  be  found  to  be  free  from  amboceptors,  whereas 
by  adding  a  little  normal  complement  serum  to  the  corpuscles  hemolysis 
results,  indicating  that  the  amboceptors  had  been  bound  to  these.  If  a 
normal  serum  contains  several  different  amboceptors  for  as  many  dif- 
ferent bloods,  all  may  be  removed  at  the  one  time  by  adding  the  respec- 
tive corpuscles  to  the  serum  and  allowing  sufficient  time  to  elapse  for 
the  amboceptors  to  become  linked  to  their  corpuscles. 

Normal  serum,  therefore,  probably  contains  numerous  antibodies  of 
the  amboceptor  type,  adapted  to  dissolve  various  foreign  substances 


NORMAL   HEMOLYSINS 


391 


when  they  gain  access  to  the  blood.  It  is  probable  that,  aside  from 
hemolysins,  various  normal  and  immune  cytolysins  play  an  important 
part  in  the  processes  of  immunity. 

While  the  normal  hemolysins  that  may  be  found  in  the  serums  of 
various  animals  havev  not  as  yet  been  fully  worked  out,  the  following 
table,  compiled  by  Sachs1  is  a  resume  of  the  work  reported  in  the  litera- 
ture on  the  subject: 

TABLE  7.— NATURAL  HEMOLYSINS 


THE 

SERU 

M    OF 

HEMOLYZE  THE 
ERYTHROCYTES 

OF    THE— 

Rabbit 

Guinea- 
pig 

! 

§ 

1 

O 

M 

O 

a 

1 

B 

O 

^4 
« 

P 

•J 

§ 

s 

a 

a> 

w 

Rabbit  

0 

+ 

4- 

4- 

4- 

4- 

4- 

4- 

± 

4- 

4- 

4- 

4- 

Guinea-pig 

4- 

o 

4- 

-f 

4- 

4. 

4- 

4. 

± 

4- 

4. 

=b 

4. 

Doe 

(+) 

=fc 

0 

Man 

± 

4- 

4- 

b 

4- 

4- 

=t 

± 

4- 

4- 

f=t) 

4- 

Goat      

4- 

4- 

4- 

0 

4- 

4- 

Ox      

=fc 

=fc 

c±) 

=t 

0 

JT 

± 

4- 

4- 

. 

= 

Sheep  
Hog  

± 
d= 

+ 

+ 

± 

=h 

0 

± 
0 

— 

4- 

+ 



4- 

Horse 

db 

-f 

4- 

4- 

4. 

± 

4. 

o 

Goose  
Duck 

+ 

4- 

0 

0 

— 

— 

Pigeon  
Hen  

+ 
+ 

+ 

efa 

+ 

— 

— 

— 

— 

0 

0 

4-  =    Well-marked  hemolysis. 

(+)=  Questionable  or  feeble  hemolysis. 

=«=  =    Doubtful. 

—  =    No  hemolysis. 

Kolmer  and  Casselman2have  titrated  the  natural  hemolysins  for  the 
corpuscles  of  various  vertebrates  in  a  large  number  of  human  serums. 
Most  interest  centers  about  the  occurrence  of  natural  antisheep  hemoly- 
sin,  because  of  the  wide-spread  use  of  an  antisheep  hemolytic  system  in 
complement-fixation  reactions,  as,  for  example,  the  Wassermann  syphilis 
reaction.  In  over  80  per  cent,  of  human  serums  there  is  present  suf- 
ficient natural  amboceptor  for  sheep's  cells  to  give  well-marked  or  com- 
plete hemolysis.  Although  this  factor  must  be  considered  in  using  an 
antisheep  hemolytic  system  in  complement-fixation  reactions,  yet  with  a 
proper  understanding  of  principles  and  the  employment  of  a  satisfac- 
tory technic  the  danger  of  error  is  reduced  to  a  minimum.  In  the  fol- 
lowing table  the  percentages  of  serums  showing  100,  75,  25,  and  0  per 

1  Sachs,  H. :    Handbuch  der  pathogenen  Mikroorganismen,  Kolle  and  Wasser- 
mann, 2.  Auflage,  2,  p.  799. 

2  Jour.  Infect.  Dis.,  1915,  16,  441. 


392 


HEMOLYSINS 


cent,  hemolysis,  with  the  maximum  dose  of  serum  (0.2  c.c.)  used  in  the 
method  of  titration,  are  shown: 

TABLE  8.— SUMMARY  OF  NATURAL  HEMOLYSINS  IN  HUMAN  SERUM 


BLOOD 

NUMBER  OF 
SERUMS 
TESTED 

PER  CENT. 
SHOWING 
100  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING 
75  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING 
25  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING  NO 
HEMOLYSIS 

Sheep  

125 

64 

20 

9 

75 

Dog  .  .  -, 

25 

16 

36 

30 

18 

Ox  

Goat 

85 
25 

6 

o 

20 

8 

24 
16 

50 
76 

Hog  
Rat   .    .    . 

40 
25 

0 
0 

1 

o 

3 

12 

96 

88 

Chicken.  .  .  . 
Horse  .  . 

25 
25 

0 
0 

0 

o 

8 
4 

92 
96 

Rabbit  

25 

o 

o 

4 

96 

Guinea-pig  . 

50 

0 

0 

2 

98 

Kolmer  and  Williams 1  have  likewise  studied  the  hemolysins  found 
in  normal  rabbit  serum,  as  preliminary  to  some  work  concerning  the  site 
of  formation  of  immune  hemolysins.  The  results  obtained  with  the 
maximum  dose  of  serum  (0.2  c.c.)  are  given  in  the  following  table: 

TABLE  9.— SUMMARY  OF  NATURAL  HEMOLYSINS  IN   NORMAL 
RABBIT  SERUM 


BLOOD 

NUMBER  OF 
SERUMS 
TESTED 

PER  CENT. 
SHOWING 
100  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING 
75  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING 
25  PER  CENT. 
HEMOLYSIS 

PER  CENT. 
SHOWING  NO 
HEMOLYSIS 

Goat  

25 

4 

52       I 

88 

12 

Sheep  

50 

18 

56 

76 

24 

Dog  

25 

32 

40 

72 

28 

Human  .... 

50 

0 

4 

20 

80 

Hog 

25 

0 

4 

20 

80 

Ox  

25 

0 

4 

20 

80 

Chicken  .... 

25 

0 

4 

8 

92 

Guinea-pig  . 
Rat,  white  .  . 

25 
25 

0 
0 

0 
0 

4 
0 

96 
100 

ISOHEMOLYSINS 

In  1892  Maragliano2  directed  attention  to  the  fact  that  the  blood- 
serum  of  patients  afflicted  with  various  diseases  exerted  a  hemolytic 
influence  on  the  blood-corpuscles  of  healthy  persons.  Later  Ascoli3 

1  Jour.  Infect.  Dis.,  1913,  xiii,  No.  1,  96. 

2  Deutsch.  med.  Wochenschr.,  1892,  xviii,  411. 

3  Munch,  med.  Wochenschr.,  1901,  xlviii,  1239. 


ISOHEMOLYSINS  393 

found  the  serums  of  cancer,  pneumonia,  and  Addison's  disease  to  be 
actively  hemolytic  for  normal  corpuscles.  Weil,  Crile,  Blumgarten 
and  Whittemore,  and  others  have  found  a  large  proportion  of  the  serums 
of  lower  animals  and  man  suffering  with  malignant  tumors  to  possess 
hemolytic  activity  for  normal  erythrocytes,  and  that  a  reaction  based 
upon  this  property  may  have  diagnostic  value.  While  isohemolysins 
are  to  be  especially  found  in  the  serums  of  cancerous  persons,  and  the 
corpuscles  of  tuberculous  persons  are  hypersensitive  and  readily  hemo- 
lyzed,  reactions  based  upon  these  observations  are  not  specific,  although 
they  may  have  some  diagnostic  value  in  relation  to  other  symptoms. 
It  is  well  to  remember  this  property  of  cancer  serum,  especially  in  making  / 
blood  transfusions. 

The  results  of  many  isohemolytic  tests  with  human  sera  and  cor- 
puscles by  different  investigators  have  shown  that  human  isohemolysins 
may  be  present,  and  that  they  have  a  practical  importance  in  relation 
to  the  dangers  of  blood  transfusion.  The  consensus  of  opinion  is  to  the 
effect,  however,  that  hemagglutinins  are  of  more  importance  in  this 
connection.  The  technic  of  these  tests  is  given  on  page  311. 

Production  of  Immune  Hemolysins. — Hemolysins  are  readily  pro- 
duced by  injecting  suitable  animals  with  several  doses  of  red  blood- 
corpuscles.  For  this  purpose  rabbits  are  commonly  employed.  Some 
hemolysins  are  more  readily  produced  than  others;  for  example,  anti- 
sheep  hemolysin  is  easily  prepared,  whereas  it  is  far  more  difficult  to 
secure  a  potent  antihuman  hemolysin.  The  various  methods  employed 
in  preparing  hemolytic  serums  have  been  described  in  the  chapter  on 
Active  Immunization  of  Animals.  Antisheep  hemolysin  for  conducting 
the  Wassermann  reaction  is  readily  prepared  by  giving  a  rabbit  three  or 
four  intravenous  injections  of  5  c.c.  of  a  10  per  cent,  suspension  of  washed 
sheep's  cells  in  sterile  normal  salt  solution  at  intervals  of  three  days. 
The  blood-cells  should  always  be  washed  three  or  four  times  with  an 
excess  of  salt  solution  to  remove  all  traces  of  serum,  in  order  that  pre- 
cipitins  may  not  be  produced  and  anaphylactic  shock  of  the  inoculated 
animal  avoided. 

The  portion  of  the  erythrocyte  that  is  responsible  for  the  production 
of  hemolysins  is  a  moot  question.  Bordet  and  von  Dungern  maintain 
that  the  stroma  is  the  exciting  agent;  Nolf  and  others  believe  that  the 
stromata  produce  hemagglutinins,  and  that  the  hemoglobin  is  chiefly 
concerned  in  the  production  of  the  hemolysin. 

General  Properties  of  Hemolysins. — Hemolysins  are  highly  resistant 
antibodies,  and  are  easily  preserved.  Sterile  immune  serum  may  be 
inactivated  by  heating  in  a  water-bath  for  half  an  hour  at  56°  C.,  and 


394  HEMOLYSINS 

may  be  preserved  for  many  months  if  small  amounts  are  placed  in  am- 
pules and  kept  in  a  cold  place.  If  an  equal  quantity  of  neutral  glycerin 
is  added  to  the  clear  inactivated  serum  it  will  aid  greatly  in  its  preserva- 
tion. The  amboceptors  resist  drying  to  a  well-marked  degree,  and  filter- 
paper  saturated  with  the  immune  serum  and  dried,  after  the  method  of 
Noguchi,  preserves  the  hemolytic  activity  in  a  remarkable  degree. 
Heating  an  immune  serum  at  56°  C.,  for  half  an  hour,  as  in  the  process 
of  inactivating  complement,  does  not  materially  injure  the  hemqlytic 
activity  of  a  potent  serum.  A  temperature  of  70°  C.  or  above  may 
cause  deterioration  and  finally  destroy  the  amboceptors. 

As  was  previously  mentioned,  hemolytic  amboceptors  possess  a 
great  affinity  for  the  receptors  of  their  homologous  corpuscles,  and  will 
readily  unite  with  them  at  a  low  temperature.  At  incubator  temperature 
the  union  is  quite  rapid,  so  that  corpuscles  may  be  " sensitized"  within 
half  an  hour. 

Source  of  Hemolysins. — As  has  been  stated  elsewhere,  MetchnikofT 
regards  the  leukocytes  as  the  source  of  fixateur  or  amboceptor  forma- 
tion. Bulloch  found  that  the  amount  of  hemolytic  amboceptor  in  a 
serum  runs  parallel  with  the  number  of  mononuclear  leukocytes,  and  he 
regards  this  as  an  indication  of  the  activity  of  the  lymphoid  tissue  in 
general,  which  he  considers  as  the  main  source  of  amboceptor  formation. 
While  it  is  probable  that  endothelial  cells  and  mononuclear  leukocytes 
are  especially  concerned  in  the  process,  our  own  investigations  in  this 
field  would  indicate  that  the  process  is  more  general,  being  participated 
in  by  cells  of  other  tissues  which  possess  suitable  combining  affinities 
for  the  alien  corpuscles. 

Antihemolysins. — A  further  step  in  the  study  of  hemolysins,  but  one 
more  of  theoretic  than  of  practical  interest,  was  the  discovery  of  anti- 
hemolysins.  By  injecting  guinea-pigs  with  normal  rabbit  serum  con- 
taining amboceptors  for  ox  blood,  Bordet  secured  a  serum  that  inhibited 
the  action  of  anti-ox  immune  serum.  Ehrlich  and  Sacks,  by  injecting  a 
goat  with  normal  rabbit  serum,  likewise  secured  a  serum  that  acted  as 
an  anti-amboceptor  against  immune  hemolytic  amboceptors  for  ox 
blood.  Ehrlich  argued  that  the  anti-amboceptor  acted  against  the  com- 
plementophile  group  of  the  amboceptor,  which  prevented  union  with  a 
complement  from  taking  place.  This  view  was  advanced  in  support  of 
his  theory  concerning,  the  two-armed  character  of  the  amboceptor  and 
that  an  anti-amboceptor  may  be  produced  against  either  the  cytophile 
or  the  complementophile  group  or  both.  In  these  particular  serums, 
however,  the  investigators  may  have  been  working  with  an  anticomple- 
ment  instead  of  an  anti-amboceptor. 


PRACTICAL   APPLICATIONS  395 

A  specific  hemolysin — one,  for  example,  specific  for  dog  blood,  de- 
rived from  treating  a  rabbit  with  dog  cells — is  highly  toxic  for  dogs,  being 
capable  of  producing  hemolysis  in  vitro  and  a  clinical  condition  known 
as  hemolytic  jaundice.  It  is  possible,  however,  gradually  to  immunize 
a  dog  against  this  amboceptor  for  his  own  cells  by  starting  with  very 
small  doses  and  gradually  increasing  these  until  it  is  found  that  the 
animal  tolerates  amounts  that  would  be  fatal  to  non-immunized  animals. 
If  a  portion  of  this  serum  is  now  added  to  the  specific  hemolytic  serum, 
it  will  be  found  that  the  power  of  the  latter  is  inhibited.  Although  this 
action  may  likewise  be  due  to  anticomplement,  it  is  probable  that  an 
anti-amboceptor  against  the  cytophile  group  of  the  amboceptor  is  also 
formed,  which  prevents  the  amboceptor  from  uniting  with  the  red  blood- 
cells,  although  conclusive  experimental  evidence  of  this  has  not  been 
adduced. 

PRACTICAL  APPLICATIONS 

There  is  probably  no  other  group  of  antibodies  that  possesses  greater 
diagnostic  value  than  do  the  hemolysins,  a  fact  that  was  demonstrated 
in  the  practical  application  of  the  Bordet-Gengou  phenomenon  of  com- 
plement fixation  in  the  diagnosis  of  syphilis  and  other  infections.  By 
adding  sensitized  corpuscles — i.  e.,  corpuscles  with  their  homologous  am- 
boceptors — to  a  fluid,  the  presence  or  absence  of  complement  may  be 
determined.  If  complement  is  present,  hemolysis  will  occur  and  be 
complete  or  partial,  depending  upon  the  amount  of  complement  avail- 
able; if  hemolysis  does  not  occur,  it  may  be  concluded  that  free  com- 
plement is  absent.  This  is  the  basis  of  the  complement-fixation  diag- 
nosis of  syphilis,  gonorrhea,  glanders,  differentiation  of  proteins,  etc. 
When  a  proper  amount  of  complement  is  added  to  a  mixture  of  antigen 
and  its  immune  serum  (containing  amboceptors),  it  is  bound  to  these 
amboceptors,  so  that  when  corpuscles  and  hemolytic  amboceptor  are 
subsequently  added,  hemolysis  does  not  occur,  since  there  is  no  available 
complement,  it  having  been  "fixed"  by  the  first  amboceptors.  If,  how- 
ever, amboceptors  for  the  antigen  in  the  first  instance  are  not  present, 
as  where  a  normal  serum  is  used,  the  complement  remains  free  and 
acts  with  the  hemolytic  amboceptor  to  produce  hemolysis  of  the  test 
corpuscles.  In  this  manner  the  hemolysins  and  their  corresponding 
corpuscles  are  employed  as  indicators  or  tests  for  the  presence  of  free 
complement,  so  that  if  an  antigen  is  known,  the  antibody  may  be  deter- 
mined; or  vice  versa,  by  using  a  known  antibody,  the  antigen  may  be 
determined,  the  criterion  in  each  instance  being  whether  complement  is 
or  is  not  bound  or  "fixed,"  a  fact  that  is  determined  by  the  subsequent 
addition  of  a  hemolysin  and  its  homologous  corpuscles. 


396  HEMOLYSINS 

The  practical  applications  and  technic  of  these  reactions  are  given 
in  subsequent  chapters. 

The  hemolysins  have  no  therapeutic  application  or  value.  They 
have  their  chief  value  in  diagnostic  reactions  and  in  the  study  of  cyto- 
lytic  phenomena  in  general. 

Quantitative  Relationship  between  Hemolytic  Amboceptor  and 
Complement. — From  a  practical  as  well  as  a  theoretic  standpoint  an 
important  property  of  amboceptor  and  of  complement  is  the  quanti- 
tative relationship  that  each  bears  to  the  other.  This  is  especially  im- 
portant in  hemolytic  reactions,  where  an  excess  of  either  may  compen- 
sate for  a  decrease  of  the  other  and  yield  fallacious  results.  If  a  certain 
amount  of  guinea-pig's  complement  is  necessary  to  lyse  1  c.c.  of  a  2.5 
per  cent,  suspension  of  sheep's  cells,  along  with  hemolytic  amboceptor, 
then  double  this  amount  of  complement  will  be  required  to  lyse  2  c.c. 
of  the  same  blood,  and  so  on.  If  a  constant  quantity  of  corpuscles  and 
hemolysin  are  added  to  a  series  of  test-tubes,  and  increasing  amounts  of 
complement  after  incubating  the  mixtures  for  an  hour  the  smallest 
amount  of  complement  that  produces  complete  hemolysis  is  called  a 
unit,  and  in  this  manner  the  strength  or  activity  of  a  serum  complement 
is  measured  or  titrated.  In  a  similar  manner  the  hemolytic  activity  of  a 
serum  or  its  measure  of  hemolysin  may  be  determined  by  placing  in  a 
series  of  tubes,  as  previously  directed,  a  definite  and  equal  amount  of 
corpuscle  suspension,  and  to  each  tube  is  then  added  an  amount,  also 
definite  and  equal,  of  a  normal  serum  as  complement,  which  is  knowi\ 
to  be  incapable  of  causing  hemolysis.  There  are  next  added  decreasing 
and  graduated  amounts  of  the  immune  serum  whose  native  complement 
has  been  destroyed  by  inactivation.  After  incubating  the  mixtures  for 
an  hour,  the  smallest  amount  of  inactivated  immune  serum  that  will  just 
produce  complete  hemolysis  is  known  as  the  amboceptor  unit  of  the  serum. 
In  other  words,  there  are  three  substances  concerned  in  serum  hemolysis : 
the  amboceptor,  the  corpuscles,  and  the  complement.  By  taking  two 
of  these  as  constants,  e.  g.,  the  corpuscles  and  the  complement,  the  unit 
of  amboceptor  may  be  determined;  or  by  taking  the  corpuscles  and 
amboceptor  as  constants  the  unit  of  complement  may  be  determined. 
Since  the  corpuscles  and  amboceptor  are  most  stable,  these  may  be 
used  as  constants,  and  the  unit  of  complement  determined  under  these 
conditions  as  preliminary  to  complement-fixation  reactions. 

It  is  important  to  bear  in  mind,  in  this  connection,  that  the  titer  of 
an  immune  hemolytic  serum  will  vary  with  the  complement  used.  For 
example,  an  antisheep  amboceptor  is  much  more  active  when  guinea-pig 


PRACTICAL   APPLICATIONS  397 

serum  is  used  as  complement  than  it  is  when  tested  with  the  same 
quantity  of  rabbit  serum  as  complement. 

After  determining  the  unit  of  amboceptor  or  complement — that  is, 
after  adjusting  the  hemolytic  system  to  exact  proportions — the  results 
that  follow  the  varying  quantities  of  complement  and  amboceptor  re- 
quire the  most  careful  consideration.  Less  than  one  unit  of  amboceptor 
with  one  unit  of  complement  cannot  yield  complete  hemolysis;  likewise 
if  with  one  unit  of  amboceptor  less  than  a  unit  of  complement  is  combined 
hemolysis  is  incomplete;  with  one  unit  of  amboceptor  and  one  unit  of 
complement  and  a  double  dose  of  corpuscles  hemolysis  will  also  be  in- 
complete. With  less  than  a  unit  of  complement  and  an  excess  of  ambo-,|/ 
ceptor,  however,  hemolysis  may  be  complete.  The  complement  may  be  * 
reduced  to  so  small  an  amount  that  hemolysis  is  incomplete  no  matter 
how  much  amboceptor  is  used,  but  the  important  fact  to  be  borne  in 
mind  is  that  a  slight  decrease  in  complement  may  be  compensated  for 
by  the  presence  of  many  units  of  amboceptor,  so  that  complete  hemolysis 
results  and  a  false  reaction  is  secured.  The  converse  of  this  is  true  to  a 
less  marked  extent — i.  e.,  an  excess  of  complement  may  compensate  for 
a  decrease  in  amboceptor,  but  is  less  capable  of  doing  so. 

These  facts  are  of  the  utmost  importance  in  making  hemolytic  ex-  '• 
periments,  as  in  complement-fixation  reactions,  where  the  entire  test 
depends  upon  demonstrating  whether  or  not  a  portion  or  the  whole  of 
the  complement  used  has  been  fixed.  Unless,  in  a  series  of  hemolytic 
reactions,  the  amount  of  amboceptor  employed  is  the  same  throughout, 
the  amount  of  complement  acting  in  these  cannot  be  determined  by 
comparing  the  degree  of  hemolysis.  This  is  true  especially  in  cases 
where  a  small  amount  of  complement  is  fixed,  as  in  the  Wassermann  re- 
action, with  a  serum  containing  a  small  amount  of  syphilitic  antibody, 
when  the  presence  of  an  excess  of  hemolytic  amboceptor  may  give 
complete  hemolysis  and  overshadow  the  fact  that  a  small  amount  of 
complement  has  been  actually  fixed  by  syphilitic  antibody  and  antigen. 

Method  of  Titration  of  Immune  Hemolysin. — Various  methods  have 
been  employed  by  different  workers  in  this  field,  but  all  are  based  upon 
the  same  principles  as  have  been  here  outlined. 

A  small  amount  of  immune  serum  is  inactivated  by  heating  in  a  water- 
bath  at  56°  C.  for  half  an  hour.  In  testing  the  serum  of  a  rabbit  during 
the  process  of  immunization  2  or  3  c.c.  of  blood  are  easily  secured  from 
the  ear,  and  the  serum  is  separated.  After  it  has  been  inactivated,  the 
serum  is  diluted  to  1  :  100  (1  c.c.  of  serum  to  99  c.c.  of  salt  solution,  or 
0.1  c.c.  serum+9.9  c.c.  of  salt  solution). 

Fresh  guinea-pig  serum  is  secured  for  complement  by  bleeding  a 


398 


HEMOLYSINS 


healthy  pig  under  ether  anesthesia  into  a  Petri  dish  or  centrifuge  tube. 
This  serum  is  diluted  1  : 20,  making  a  5  per  cent,  solution,  by  adding  1 
c.c.  of  serum  to  19  c.c.  of  salt  solution.  Each  cubic  centimeter  of  this 
dilution  contains  0.05  c.c.  of  undiluted  serum,  which  experience  has 
shown  is  a  satisfactory  amount  to  use. 

The  corpuscle  suspension  is  then  prepared.  The  blood  used  depends 
upon  the  kind  of  amboceptor  that  is  to  be  titrated.  With  sheep  and  ox 
blood,  a  2.5  per  cent,  suspension  of  washed  corpuscles  may  be  employed. 
With  antihuman  amboceptor,  the  corpuscles  are  usually  used  in  1  per 
cent,  suspension.  (See  Noguchi  Modification  of  Wassermann  Reac- 
tion.) After  the  corpuscles  have  been  washed  three  times,  1  c.c.  is 
placed  in  39  c.c.  of  salt  solution,  or  sufficient  salt  solution  is  added  to 
2.5  c.c.  of  the  corpuscles  to  make  the  total  volume  equal  100  c.c. 

To  a  series  of  six  sterile  test-tubes  increasing  doses  of  the  diluted 
immune  serum  are  now  added,  together  with  1  c.c.  of  complement  dilu- 
tion, 1  c.c.  of  corpuscles  suspension,  and  sufficient  normal  salt  solution 
to  make  the  total  volume  in  each  tube  about  3  or  4  c.c.  The  follow- 
ing table  shows  the  method  of  preliminary  titration  of  a  hemolytic 
serum: 


TABLE  10.— PRELIMINARY  TITRATION  OF  A  HEMOLYSIN 


AMOUNT  OF  INACTIVATED 

DOSE  OF 

DOSE  OF 

CORPUS- 

NORMAL 

RESULT  OF  HEMOLYSIS 

TUBE 

IMMUNE  SERUM  IN  C.c. 

CLES,  C.C. 

SALT 

AFTER  ONE  HOUR  IN  THE 

(1:100) 

c.  (1:20) 

(2.5pEn 

CENT.) 

SOLUTION 

INCUBATOR  (37°  C.) 

1.  . 

0.1  (0.001  c.c.  undiluted) 

1 

q.  s.  3  c  c 

No  hemolysis 

2  

0.2  (0.002  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Partial  hemolvsis 

3 

0.4  (0.004  c.c.  undiluted) 

1 

q  s   3  c  c 

Complete  hemolysis 

4  

0.6  (0.006  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolvsis 

5  

0.8  (0.008  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

6  

1.0  (0.01    c.c.  undiluted) 

1 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

In  this  instance  the  unit  of  amboceptor  is  1  : 250,  which  is  too  low 
for  a  satisfactory  antisheep  serum.  The  rabbit  should,  therefore, 
receive  another  dose  or  two  of  corpuscles,  and  the  serum  be  titrated  again 
in  from  four  to  seven  days  after  the  last  injection  has  been  given.  In 
this  titration  it  will  be  well  to  use  a  higher  dilution  of  the  inactivated 
immune  serum,  as  1  : 1000.  This  may  be  prepared  by  adding  1  c.c.  of 
a  dilution  of  1  : 100  with  9  c.c.  of  normal  salt  solution  and  mixing  well. 
The  titration  is  then  proceeded  with  as  follows: 


FIG.  106. — TITRATION  OF  HEMOLYTIC  AMBOCEPTOR. 

The  tube  containing  0.3  c.c.  hemolytic  amboceptor  is  the  smallest  amount  showing 
complete  hemolysis,  and  this  amount  is  the  unit. 


PRACTICAL  APPLICATIONS  399 

TABLE  11.— TITRATION  OF  HEMOLYSIN 


DOSE 

DOSE 

OF 

OF 

COR- 

AMOUNT OF  INACTIVATED 

COM- 

PUS- 

NORMAL 

RESULT  OF  HEMOLYSIS 

TUBE 

IMMUNE  SERUM  IN  C.c. 

PLE- 

CLES, 

SALT 

AFTER  ONE  HOUR  IN  THE 

(1  :  1000) 

MENT 

C.c. 

SOLUTION 

INCUBATOR  AT  37°  C. 

INC.C. 

(2.5 

(1:20) 

PER 

CENT.) 

1 

0.05  (0.00005  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

No  hemolysis 

2     . 

0.1    (0.0001    c.c.  undiluted) 

1 

q.  s.  3  c.c. 

No  hemolysis 

3     . 

0.15  (0.00015  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Beginning  hemolysis 

4     . 

0.2    (0.0002    c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Partial  hemolysis 

5     . 

0.25  (0.00025  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Just  complete  hemolysis 

6|  . 

0.3    (0.0003    c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

7    . 

0.35  (0.00035  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

8     . 

0.4    (0.0004    c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

9     . 

0.45  (0.00045  c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

10     . 

0.5    (0.0005   c.c.  undiluted) 

1 

q.  s.  3  c.c. 

Complete  hemolysis 

The  following  controls  should  be  set  up  at  the  same  time : 

1.  1  c.c.  of  corpuscles  in  1  c.c.  of  amboceptor  dilution.     This  tube 

should  show  no  hemolysis,  as  the  serum  has  been  inactivated 
and  is  too  highly  diluted  for  complement  activity,  even  though 
native  complement  were  present. 

2.  1  c.c.  of  corpuscles  in  1  c.c.  of  complement  dilution.     This  tube 

may  show  a  trace  of  hemolysis,  due  to  the  presence  of  a  small 
amount  of  natural  amboceptor  for  the  corpuscles  used.  As  a 
general  rule,  guinea-pig  serum  is  free  from  natural  antisheep 
amboceptor,  or  the  amount  is  so  small  under  these  conditions 
that  it  is  not  necessary  to  remove  it. 

.    3.  1  c.c.  of  corpuscles  in  3  c.c.  of  salt  solution.   This  tube  should  show 
no  hemolysis,  and  serves  to  show  that  the  diluent  was  isotonic. 
In  the  foregoing  titration  it  is  found  that  0.25  c.c.  of  1  :  1000  dilution 
of  amboceptor  is  the  unit,  or  the  titer  is  1  :  4000.     This  method  is  less 
difficult  than  preparing  a  series  of  dilutions  of  amboceptor  as  a  1  :  100, 
1  :  200,  1  :  500,  1  :  1000,  1  :  2000,  etc.,  using  a  cubic  centimeter  of  each 
dilution.    The  method  requires  accurate  pipets  and  careful  work,  but 
yields  uniform  and  satisfactory  results  (Fig.  106). 

The  rabbit  may  now  be  bled  under  anesthesia.  The  serum  is  sep- 
arated and  inactivated  and  again  titrated,  as  the  final  titration,  for  some 
unknown  reason,  is  likely  to  be  a  little  lower  than  in  the  primary  tests. 
When  suitably  preserved,  a  hemolytic  serum  will  maintain  its  activ- 
ity for  long  periods  of  time;  it  should  always,  however,  be  titrated  before 
complement-fixation  tests  are  undertaken. 


400  HEMOLYSINS 

Methods  for  Removing  Hemolysins  from  a  Serum. — In  general, 
these  aim  to  remove  the  natural  hemolysins,  such  as  natural  antisheep 
hemolysin,  from  human  serums  preliminary  to  making  the  Wassermann 
test,  or  from  a  guinea-pig  serum  that  is  to  be  used  as  complement. 

The  method  of  removal  consists  simply  in  adding  corpuscles  to  the 
serum,  and  allowing  sufficient  time  for  the  corresponding  hemolytic 
amboceptor  to  become  attached  and  then  removing  both  by  centrifuging 
the  mixture.  If  the  serum  is  fresh,  it  should  be  cooled  to  0°  to  3°  C., 
in  order  to  inhibit  complement  activity,  which  would  hemolyze  a  por- 
tion of  the  corpuscles. 

To  remove  natural  antisheep  hemolysin  from  a  patient's  serum  place 
a  measured  quantity  of  cold  serum  in  a  centrifuge  tube  and  add  four 
volumes  of  a  2.5  per  cent,  suspension  of  sheep's  cells.  This  will  make  a 
dilution  of  1  :  5,  so  that  0.5  c.c.  of  the  dilution  is  equivalent  to  0.1  c.c.  of 
undiluted  serum.  After  placing  the  tube  in  ice-water  for  from  fifteen 
to  thirty  minutes  centrifuge  thoroughly  to  remove  the  corpuscles.  The 
process  may  be  carried  out  after  the  serum  has  been  inactivated,  in 
which  case  it  is  not  necessary  to  work  with  cold  serum. 

In  removing  a  natural  hemolytic  amboceptor  from  a  guinea-pig 
serum  that  is  to  be  used  as  complement  a  measured  amount  of  serum  is 
first  removed  to  a  separate  tube  and  thoroughly  chilled  in  a  glass  of 
cracked  ice.  If  a  large  amount  of  serum  is  to  be  used,  for  example,  5  c.c., 
it  is  well  to  place  about  0.1  to  0.2  c.c.  of  pure  undiluted  corpuscles,  after 
their  last  washing,  in  the  bottom  of  a  centrifuge  tube.  This  quantity 
of  corpuscles  does  not  materially  affect  the  dilution  of  the  serum.  If  a 
smaller  amount  of  serum  is  used,  such  as  1  c.c.,  it  is  well  to  add  8  c.c.  of  a 
2.5  per  cent,  suspension  of  corpuscles,  and  after  centrifuging  the  mixture 
the  final  dilution  of  1  :  20  is  secured  by  adding  10  c.c.  of  salt  solution  to 
the  supernatant  diluted  serum. 

Method  of  Determining  Natural  Hemolysins  in  Serum. — To  ascer- 
tain whether  or  not  a  certain  natural  amboceptor  is  present  in  a  serum  it 
is  merely  necessary  to  inactivate  the  serum,  and  to  a  measured  amount, 
for  example,  0.2  c.c.,  add  1  c.c.  of  complement  serum  (1  :  20)  that  is 
known  to  be  free  of  the  particular  amboceptor  in  question,  and  1  c.c. 
of  a  2.5  per  cent,  suspension  of  the  corresponding  corpuscles.  Sufficient 
salt  solution  is  added  to  bring  the  total  volume  to  3  or  4  c.c.  The  mix- 
ture is  then  incubated  at  37°  C.  for  one  or  two  hours,  when  the  occur- 
rence of  hemolysis  indicates  the  presence  of  the  amboceptor  for  the 
corpuscles  employed. 

To  determine  the  amount  of  natural  amboceptor  by  titration  dilute 


SERUM    DIAGNOSIS   OF   PAROXYSMAL   HEMOGLOBINURIA       401 

the  serum  with  nine  parts  of  normal  salt  solution,  and  to  a  series  of  test- 
tubes  add  increasing  amounts  of  0.05  c.c.,  0.1  c.c.,  0.2  c.c.,  0.4  c.c.,  0.8 
c.c.,  1  c.c.,  and  2  c.c.,  corresponding  respectively  to  0.005,  0.01,  0.02, 
0.04,  0.08,  0.1,  and  0,2  c.c.  of  the  undiluted  serum.  Add  1  c.c.  of  a  5 
per  cent,  dilution  of  fresh  amboceptor-free  guinea-pig  serum  as  com- 
plement, and  1  c.c.  of  a  2.5  per  cent,  suspension  of  the  corpuscles; 
sufficient  salt  solution  is  added  to  make  the  total  volume  about  4  c.c. 
After  shaking,  the  tubes  are  placed  in  the  incubator  at  37°  C.  for  two 
hours,  removed,  and  the  results  read,  or  the  tubes  may  be  placed  in  a 
refrigerator  overnight  and  the  results  read  in  the  morning. 


SERUM  DIAGNOSIS  OF  PAROXYSMAL  HEMOGLOBINURIA 

A  hemolytic  substance  may  be  demonstrated  in  the  blood-serum  of 
most  cases  of  paroxysmal  hemoglobinuria  at  certain  periods.  Although 
the  exact  nature  of  this  substance  is  unknown,  it  has  many  of  the  prop- 
erties of  isohemolysins,  being  capable  of  sensitizing  the  red  corpuscles 
of  the  patient  or  those  of  a  normal  person  at  a  low  temperature,  hemoly- 
sis  being  effected  in  the  presence  of  fresh  serum,  presumably  with  com- 
plement, and  best  at  body  temperature. 

According  to  Cook,  about  90  per  cent,  of  hemoglobinurics  show  a 
positive  Wassermann  reaction.  Landsteiner  found  that  about  10 
per  cent,  of  paretics  showed  similar  reactions,  and, other  observers  have 
reported  finding  isohemolysins  in  epileptics  and  idiots.  Malaria  and 
trypanosomiasis  have  also  been  regarded  as  causes  of  this  condition,  the 
most  evidence,  however,  indicating  that  the  etiology  has  a  luetic  origin. 
It  is  possible  that  the  hemotoxin  is  similar  to  the  hemolysin  of  cobra 
venom,  being  in  the  nature  of  an  amboceptor  complemented  by  the 
fatty  acids  or  lecithin  of  red  corpuscles  (endocomplement)  or  by  a  serum 
complement. 

First  Method. — According  to  Ehrlich,  a  small  tourniquet  should  be 
applied  about  the  base  of  one  of  the  patient's  fingers,  and  this  is  then 
kept  immersed  in  ice-cold  water  for  half  an  hour.  Blood  from  the  finger 
thus  constricted  is  then  collected  in  a  Wright  capsule,  and  blood  from  a 
finger  of  the  other  hand  is  used  as  a  control.  Both  are  allowed  to  clot 
and  are  then  centrifugalized.  The  serum  from  the  finger  held  in  iced 
water  is  tinged  red  from  dissolved  hemoglobin,  whereas  the  control 
serum  is  not  tinged  or  at  least  not  tinged  so  deeply. 

Second  Method. — Donath  and  Landsteiner  have  applied  Ehrlich's 
method  in  vitro.     Their  method  consists  of  collecting  blood  in  a  small 
26 


402  HEMOLYSINS 

test-tube,  cooling  to  0°  C.  for  half  an  hour,  heating  subsequently  to 
37°  C.  for  three  hours.  The  presence  or  absence  of  hemolysis  is  observed, 
and  the  results  compared  with  those  obtained  from  normal  blood 
treated  in  the  same  manner  and  at  the  same  time. 

Third  Method. — This  technic  is  carried  out  in  vitro  in  the  following 
manner:  Pipet  2  c.c.  of  the  patient's  blood  in  a  small  test-tube  and 
separate  the  serum.  At  the  same  time  place  1  c.c.  of  blood  in  a  centri- 
fuge tube  containing  9  c.c.  of  a  1  per  cent,  solution  of  sodium  citrate  in 
normal  salt  solution.  Wash  the  corpuscles  twice  and  suspend  the  sedi- 
ment in  10  c.c.  of  normal  salt  solution.  Then,  secure  a  cubic  centimeter 
of  a  fresh  serum  from  a  normal  person.  Proceed  to  make  the  following 
mixtures : 

Tube  1 :   0.2  c.c.  patient's  serum  +  1  c.c.  corpuscle  suspension. 

Tube  2:   0.1  c.c.' patient's  serum  +  1  c.c.  corpuscle  suspension. 

Tube  3:    0.2  c.c.  normal  serum  +  1  c.c.  corpuscle  suspension. 

Tube  4:    0.1  c.c.  normal  serum  +  1  c.c.  corpuscle  suspension. 

Tube  5:   1.0  c.c.  corpuscle  suspension. 

Add  sufficient  normal  salt  solution  to  each  tube  to  make  the  total 
volume  measure  2  c.c.  Shake  gently,  and  place  in  the  refrigerator  at  a 
low  temperature  (not  higher  than  4°  C.)  for  an  hour.  Shake  each  tube 
gently  and  place  them  in  the  incubator  at  37°  C.  for  two  hours.  The 
tubes  are  then  centrifuged  and  the  presence  or  absence  of  hemolysis 
is  noted.  Usually  the  patient's  serum  shows  hemolysis  of  greater  or 
less  degree. 

Similar  mixtures  may  be  made  with  the  patient's  serum  and  the  cor- 
puscles of  a  normal  person.  The  hemolytic  substance  is  capable  of  lysing 
these  to  the  same  degree  that  it  does  the  patient's  own  cells. 

METHOD  OF  DETERMINING  THE  RESISTANCE  OF  RED  BLOOD-CORPUSCLES. 
NON-SPECIFIC  HEMOLYSIS 

Various  substances  have  been  employed  to  test  the  resistance  of  the 
red  blood-corpuscles.  Of  these,  the  hypotonic  solutions  of  sodium 
chlorid,  of  varying  strength,  have  yielded  results  of  clinical  importance, 
especially  in  the  study  of  paroxysmal  hemoglobinuria,  the  primary  ane- 
mias, etc. 

The  following  technic,  slightly  modified  after  the  methods  used  by 
Smith  and  Brown,  Gay,  Moss,  Karsner,  and  Pearce,  is  a  ready  means 
for  determining  the  resistance  of  human  corpuscles  to  salt  solutions  of 
different  tonicities.  Chemically  pure  sodium  chlorid  is  dried  for  two 
hours  at  170°  C.,  and  immediately  weighed  in  amounts  necessary  to 


SERUM    DIAGNOSIS    OF    PAROXYSMAL   HEMOGLOBINURIA       403 

make  500  c.c.  of  salt  solution,  ranging  from  0.1  to  0.6  per  cent,  in  grada- 
tions of  0.02  per  cent.  This  means  the  preparation  of  twenty-six  dif- 
ferent solutions,  which  should  be  preserved  in  proper-sized  bottles  fitted 
with  tight  rubber  stoppers. 

When  the  test  is  needed  only  occasionally,  these  solutions  are  readily 
prepared  by  filling  a  50  c.c.  buret,  graduated  in  one-tenths,  with  dis- 
tilled water,  and  another  with  a  1  per  cent,  solution  of  pure  dried  sodium 
chlorid.  From  these,  the  various  solutions  are  readily  prepared  after 
the  following  manner: 

10  c.c.  of  0.6    per  cent,  sodium  chlorid  =  6  c.c.  of  1  per  cent,  salt 

solution  -f  4  c.c.  of 
distilled  water. 

10  c.c.  of  0.58  per  cent,  sodium  chlorid  =  5.8  c.c.  of  1  per  cent,  salt 

solution  +  4.2  c.c.  of 
distilled  water. 

10  c.c.  of  0.56  per  cent,  sodium  chlorid  =  5. 6  c.c.  of  1  per  cent,  salt 

solution  -|-  4.4  c.c.  of 
distilled  water. 

10  c.c.  of  0.54  per  cent,  sodium  chlorid  =  5.4  c.c.  of  1  per  cent,  salt 

solution  +  4.6  c.c.  of 
distilled  water. 

10  c.c.  of  0.52  per  cent,  sodium  chlorid  =  5. 2  c.c.  of  1  per  cent,  salt 

solution  +  4.8  c.c.  of 
distilled  water. 

10  c.c.  of  0.5  per  cent,  sodium  chlorid  =   5  c.c.  of  1  per  cent,  salt 

solution  -f-  5  c.c.  of 
distilled  water. 

Similar  dilutions  are  made,  until  the  final  dilution  is  reached.  In 
many  instances  it  may  not  be  necessary  to  use  so  large  a  number  of  dilu- 
tions, as  from  0.5  to  0.2  per  cent,  may  be  sufficient  range  to  indicate  the 
tonicity.  . 

Five  cubic  centimeters  of  blood  are  aspirated,  under  aseptic  pre- 
cautions, from  an  arm  vein  of  the  patient,  and  immediately  placed  in 
25  c.c.  of  sterile  1  per  cent,  sodium  citrate  in  0.85  per  cent,  sodium  chlorid 
to  prevent  coagulation.  The  flask  or  large  centrifuge  tube  is  well 
shaken,  and  the  mixture  is  centrifuged  at  sufficient  speed  to  throw  down 
the  corpuscles.  The  supernatant  fluid  is  drawn  off,  and  the  corpuscles 
are  washed  once  or  twice  more  with  sterile  normal  salt  solution.  After 
the  last  washing  the  supernatant  fluid  is  removed,  leaving  the  erythro- 
cytes  at  the  bottom  of  the  tube. 


404  HEMOLYSINS 

A  series  of  small  test-tubes  (10  by  1  cm.)  are  marked  appropriately 
and  placed  in  a  rack.  To  each  tube  3  c.c.  of  the  various  hypotonic  salt 
solutions  and  0.05  c.c.  of  the  red  blood-corpuscles  (about  1  drop)  are 
added.  The  salt  solution  and  corpuscles  in  each  tube  are  well  mixed, 
and  the  whole  series  is  placed  in  the  refrigerator  for  from  eighteen  to 
twenty-four  hours,  after  which  the  readings  are  made. 

The  tube  of  lowest  dilution — even  if  it  shows  but  a  trace  of  hemol- 
ysis — represents  the  point  of  initial  hemolysis  or  minimal  resistance. 
The  strength  of  salt  solution  in  which  all  the  corpuscles  are  hemolyzed 
represents  the  point  of  complete  hemolysis  or  maximal  resistance. 

Normally,  the  minimal  resistance  is  about  0.47,  and  the  maximal 
resistance  about  0.3  (Morris).  Hill1  has  found  the  points  0.457  and 
0.340  respectively,  for  normal  blood. 

1  Archiv.  Inter.  Med.,  1915,  16,  809. 


CHAPTER  XXI 
VENOM  HEMOLYSIS 

Nature  of  Venom  Hemolysis. — In  a  previous  chapter  the  statement 
was  made  that  certain  snake  poisons,  and  especially  cobra  venom,  are 
actively  hemolytic.  Flexner  and  Noguchi x  first  demonstrated  that  the 
blood-corpuscles  of  certain  species  of  animals  undergo  hemolysis  when 
a  suitable  serum  is  present,  and  believed  that  the  venom  contained  an 
amboceptor  that  was  active  with  serum  complement. 

Shortly  afterward  Kyes2  discovered  that  venom  may  hemolyze  the 
corpuscles  of  certain  animals  without  the  presence  of  serum,  and  be- 
lieved that  the  complement-like  activator  was  contained  within  the  cor- 
puscles, to  which  he  accordingly  applied  the  name  endocomplement. 

Later  Kyes2  confirmed  Calmette's  observation  that  practically  any 
serum,  when  heated  to  65°  C.  and  higher,  showed  an  increased  activity 
in  the  process  of  venom  hemolysis.  Kyes  and  Sachs 3  then  concluded  that 
endocomplement  was  not  of  the  nature  of  a  thermolabile  complement, 
but  was,  rather,  a  combination  of  lecithin  and  the  stromata  of  erythro- 
cytes. 

Kyes  later  succeeded  in  combining  cobra  venom  and  lecithin  by  shak- 
ing a  watery  solution  of  venom  with  a  solution  of  lecithin  in  ether,  form- 
ing cobra-lecithid,  which  was  found  to  be  actively  hemolytic. 

The  erythrocytes  of  various  animals  differ  in  their  susceptibility  to 
venom  hemolysis.  For  instance,  those  of  the  dog  and  guinea-pig  are 
most  susceptible  to  the  process;  those  of  the  ox,  goat  and  sheep  are  en- 
tirely refractory,  whereas  those  of  the  horse,  rabbit,  rat,  pig,  and  man 
occupy  an  intermediate  position.  Sacks  suggested  that  the  variation 
in  hemolytic  resistance  of  red  blood-cells  from  these  species  of  animals 
was  dependent  on  the  amount  of  lecithin  contained  in  the  cells.  Kyes, 
on  the  other  hand,  believes  that  since  all  erythrocytes  contain  sufficient 
lecithin  to  activate  cobra  venom,  the  varying  susceptibility  depends 
rather  on  the  availability  of  the  intracellular  lecithin  for  the  reaction, 
i.  e.,  whether  the  lecithin  in  the  cell  is  available  in  a  free  state. 

1  Jour.  Exper.  Med.,  1902,  vi,  277. 

2  Berl.  klin.  Wochenschr.,  1902,  xxxix,  886;  ibid.,  1903,  xlii,  21;  ibid.,  1903;  xlii, 
956;   Biochem.  Zeitschr.,  1907,  iv,  109;  Jour.  Infect.  Dis.,  1910,  vii,  181. 

3  Berl.  klin.  Wochenschr.,  1903,  xliii,  21,  57,  82. 

405 


406  VENOM   HEMOLYSIS 

According  to  this  theory,  therefore,  any  factor  that  modifies  the 
availability  of  the  cell  lecithin  may  modify  the  susceptibility  of  the  cells 
for  hemolysis  with  cobra  venom. 

Noguchi1  has  questioned  the  correctness  of  this  view.  He  holds 
that  although  lecithin  exists  in  the  stroma  of  all  kinds  of  corpuscles,  it  is 
not  present  in  a  form  available  for  venom  activation,  and  that  the  de- 
gree of  susceptibility  to  hemolysis  depends  chiefly  upon  the  amount  of 
ether-soluble  activators  present  in  the  cells,  as,  for  example,  fatty  acids, 
particularly  oleinic  acid,  and  their  soluble  soaps.  In  his  opinion  heating 
an  inactive  serum  to  65°  C.  and  higher  renders  it  active  with  venom, 
owing  to  the  presence  of  a  protein  compound  of  lecithin. 

A  normal  serum  may,  therefore,  contain  two  activators,  one  being 
thermolabile  and  resembling  complement  (inactivated  by  calcium  chlo- 
rid),  and  the  other  Joeing  thermostabile  and  a  protein  lecithin.  By  add- 
ing oleinic  acid  or  its  soluble  soap  to  a  non-activating  serum  the  latter  is 
rendered  highly  active  so  far  as  venom  hemolysis  is  concerned.  Hence 
while  Kyes  regards  lecithin  as  the  chief  component  of  endocellular  com- 
plement, Noguchi  regards  the  fatty  acids,  neutral  fats,  and  soluble 
soaps  as  the  active  agents. 

Other  observers  consider  the  fatty  acids  and  soap  as  indirect  activat- 
ing agents  in  venom  hemolysis,  in  that  they  possess  the  power  of  modi- 
fying the  cell  and  rendering  the  intracellular  lecithin  available  for  the 
formation  of  complete  hemolysin. 

On  the  other  hand,  in  susceptible  cells  the  union  of  cobra  venom  and 
lecithin  occurs  directly  with  the  formation  of  the  complete  hemolysin, 
Kyes'  cobra-lecithid,.  due  to  the  splitting  of  the  fatty  acid  radical  from 
the  lecithin.  Ludecke,  von  Dungern,  and  Coca  and  Manwaring  re- 
gard this  product  as  a  venom-free  lecithin  derivative,  and  not  as  a  leci- 
thin. They  prefer  to  call  the  active  principle  "cobralecithinase,"  and 
the  complete  hemolysin"  mono-fatty-acid-lecithin." 

According  to  Kyes,  the  relative  amounts  of  lecithin  and  venom  am- 
boceptor  show  quantitative  relationship  comparable  to  serum  ambo- 
ceptors  and  complements,  namely,  that,  within  certain  limits,  the  larger 
the  amount  of  venom,  the  smaller  the  amount  of  lecithin  necessary  to 
effect  hemolysis;  and,  conversely,  the  larger  the  amount  of  lecithin,  the 
smaller  the  amount  of  venom  required. 

Further  reference  to  the  intimate  relationship  that  exists  between 
lipoids  and  complements  and  hemolysis  is  also  made  in  the  discussion 
on  the  nature  of  complements,  on  p.  351. 

1  Jour.  Exp.  Med.,  1907,  ix,  436. 


VENOM   HEMOLYSIS   IN   SYPHILIS  407 

VENOM  HEMOLYSIS  IN  SYPHILIS 

The  first  application  of  venom  hemolysis  was  made  by  Weil,1  who 
found,  in  testing  the  hemolytic  powers  of  cobra  venom  with  cells  derived 
from  persons  suffering  from  different  diseases,  that  the  red  cells  of  syphil- 
itic individuals  offered  a  characteristic  resistance.  Various  explanations 
have  been  offered  for  this  phenomenon: 

1.  It  was  argued  that  the  quantity  of  red-cell  lecithin  is  actually 
diminished  in  syphilis  after  the  primary  stage  because  pallidum  toxin 
attacks  the  same  lipoidal  substances  of  tissue  cells  as  does  cobra  venom, 
in  this  way  accounting  for  the  diminished  amount  of  lecithin  that  can 
be  extracted  in  syphilis,  as  compared  with  that  obtained  from  normal 
tissues.     Accordingly,  the  increase  in  resistance  is  only  apparent,  and 
is  due  rather  to  the  fact  that  there  is  insufficient  endocomplement  for 
the  venom  amboceptor. 

2.  Another  explanation  offered  was  that  the  increased  resistance  of 
the  red  cells  of  syphilitic  persons  to  venom  hemolysis  is  due  to  the  fact 
that  pallidum  toxin  attacks  endocomplement,  and  that  the  cells  become 
specifically  immunized  to  this  deleterious  influence  in  much  the  same 
way  that  repeated  injections  of  such  a  hemolytic  agent  as  saponin  leads 
in  rabbits  to  the  production  of  red  cells,  which  show  a  marked  resistance 
to  saponin  hemolysis  but  not  to  any  other  hemolytic  agent. 

3.  Pallidum  toxin  was  believed  to  so  affect  the  lecithin  content  of  red 
cells  as  to  render  a  smaller  quantity  of  it  available  in  a  free  state  for 
union  with  the  venom  amboceptor  to  form  the  hemolysin. 

4.  Another  theory  advanced  was  that  pallidum  toxin  effects  a  dis- 
sociation of  red  cells  between  lecithin  and  cholesterin,  the  latter  sub- 
stance causing  inhibition  of  hemolysis. 

Whatever  may  be  the  true  explanation,  the  fact  has  been  quite  well 
attested  that  the  red  cells  of  a  large  percentage  of  persons  in  the  tertiary 
stage  of  syphilis  exhibit  a  characteristic  increased  resistance  to  venom 
hemolysis,  and  while  the  cobra  hemolysis  test  in  this  disease  is  of  second- 
ary importance  to  the  Wassermann  reaction  as  a  diagnostic  procedure, 
yet  it  represents  one  of  the  most  interesting  of  biologic  phenomena,  and 
may  possibly  be  employed  in  other  clinical  methods. 

TECHNIC  OF  THE  COBRA  VENOM  TESTS 

Preparation  of  Venom  Solution. — A  1  :  1000  stock  solution  of  dried 
cobra  venom  is  prepared  by  accurately  weighing  out  0.01  gram  of  dried 

1  Proc.  Soc.  Exper.  Biol.  and  Med.,  1909,  vi,  49;  ibid.,  1909,  vii,  2;  Jour.  Infect. 
Diseases,  1909,  vi,  688. 


408  VENOM    HEMOLYSIS 

pulverized  venom  and  dissolving  this  in  10  c.c.  of  normal  saline  solution 
(1  : 1000).  This  stock  dilution  is  best  preserved  in  amounts  of  1  c.c.  in 
sealed  ampules,  kept  in  the  frozen  state  in  the  ice  chest  in  a  wide-mouthed 
well-stoppered  vacuum  bottle  containing  salt  and  ice  (Schwartz). 

Each  cubic  centimeter  is  sufficient  for  making  three  tests,  so  that  the 
10  ampules  will  be  enough  for  30  tests,  or  1  gram  of  venom  for  3000  re- 
actions. Or  the  dried  venom  may  be  weighed  out  in  amounts  of 
0.0005  gram  in  test-tubes,  and  diluted,  just  before  being  used,  with  1  c.c. 
of  normal  salt  solution  (1  :  2000).  Immediately  before  the  tests  are 
conducted  subdilutions  are  prepared  of  the  stock  dilution  (1  :  1000), 
using  separate  pipets  for  each,  as  follows: 

Solution  A:   1  :  10,000  =  1  c.c.  stock  solution  +  9  c.c.  normal  saline 

solution. 
Solution  B:    1  :  15,000  =  2  c.c.  solution  A  +  1  c.c.  normal  saline 

solution. 
Solution  C:    1  : 20,000  =  1  c.c.  solution  A  +  1  c.c.  normal  saline 

solution. 
Solution  D:    1  : 30,000  =  1  c.c.  solution  B  +  1  c.c.  normal  saline 

solution. 
Solution  E:    1  :  40,000  =  1  c.c.  solution  C  +  1  c.c.  normal  saline 

solution. 

These  amounts  are  sufficient  for  making  three  tests;  if  more  tests 
are  to  be  made,  larger  amounts  of  the  various  dilutions  will  keep  fairly 
well  in  a  good  refrigerator  for  several  days,  but  it  is  always  well  to  plan 
the  work  so  that  the  exact  amount  will  be  prepared  and  no  waste  occur. 
Each  lot  of  stock  solution  should  be  tested  occasionally  with  the  cells 
of  known  normal  and  positive  persons,  to  make  certain  that  the  venom 
is  active  in  these  dilutions.  These  titrations  are  conducted  in  the 
same  manner  as  the  test. 

Preparation  of  Blood-cells. — With  a  sterile  syringe  blood  is  drawn 
from  a  vein  at  the  elbow  and  2  c.c.  placed  in  an  accurately  graduated 
centrifuge  tube  containing  5  c.c.  of  a  2  per  cent,  solution  of  sodium 
citrate  in  normal  saline  solution.  The  suspension  should  be  shaken  gently 
to  insure  mixing  and  the  prevention  of  coagulation,  but  defibrination 
by  means  of  whipping  should  never  be  practised.  The  cells  may  be  pre- 
pared at  once  or  placed  in  the  ice-chest  overnight.  Sufficient  normal 
saline  solution  is  added  to  bring  the  total  volume  to  15  c.c.  Mix  gently 
and  centrifuge  at  low  speed  until  the  supernatant  fluid  is  clear.  Draw  off 
the  fluid,  add  more  normal  saline  solution,  mix  up  the  cells,  and  centri- 
fuge again  until  clear.  Repeat  this  process  once  more  so  that  all  traces 


f    i 


t  i 

I  il 

u  u  u 


il 


••«*-»• 


FIG.  107. — VENOM  HEMOLYSIS. 

First  row  strongly  positive;  second  row  moderately  positive;  third  row  weakly 
positive;  fourth  and  fifth  rows  negative. 


VENOM   HEMOLYSIS   IN    SYPHILIS  409 

of  serum  will  be  removed.  After  completing  the  last  centrifugaliza- 
tion,  which  should  be  thorough  (ten  minutes),  the  cells  are  diluted  with 
25  volumes  of  normal  saline  solution,  which  makes  a  4  per  cent,  sus- 
pension— for  instance,  0.8  c.c.  of  corpuscles  would  require  20  c.c.  of 
diluent. 

Just  what  influence  sodium  citrate  has  upon  the  process  is  not 
known,  but  it  is  certain  that  satisfactory  results  are  seldom  obtained 
with  blood  defibrinated  by  whipping  with  rods  of  glass  beads.  Similarly, 
if  centrifuged  too  rapidly,  cells  are  broken  up  or  rendered  more  sus- 
ceptible to  the  venom  ambocepter. 

The  Test. — Into  a  series  of  five  test-tubes  (4  by  %  inches)  place 
1  c.c.  of  each  dilution  of  venom,  and  label  each  tube  correctly;  add  1  c.c. 
of  the  4  per  cent,  suspension  of  cells  to  each  tube,  shake  gently,  and  incu- 
bate for  one  hour  at  37°  C.  Except  in  cases  where  the  venom  dilutions 
are  being  frequently  used  and  are  known  to  be  reliable,  controls  should 
be  included.  I  usually  add  a  normal  control  of  cells  from  a  healthy 
person  with  the  1 : 30,000  or  1 : 40,000  dilution  and  expect  complete 
hemolysis  to  occur.  A  preliminary  reading  is  made  at  the  end  of  an 
hour;  the  tubes  are  shaken  gently  and  placed  in  the  refrigerator  over- 
night; the  final  reading  is  made  next  morning,  and  should  tally  quite 
closely  with  the  preliminary  reading. 

Reading  the  Results. — Unless  the  cells  are  derived  from  a  very  strongly 
reacting  case  of  syphilis,  the  1 :  10,000  dilution  will  be  hemolyzed.  The 
normal  control  tube  should  show  complete  hemolysis.  If  the  1 : 10,000 
tube  is  not  hemolyzed  and  some  of  the  higher  dilutions  show  hemolysis, 
an  error  in  technic  has  occurred,  and  the  test  should  be  repeated  with 
fresh  dilutions. 

The  reactions  are  read  and  recorded  as  follows  (Fig.  107) : 

ABODE 

H.     M.  H.      —  —     =  strongly  positive. 

H.       H.       M.  H.  =  moderately  positive. 

H.       H.          H.      S.  H.  =  weakly  positive. 

H.       H.          H.         H.      M.  H.  =  negative. 

H.       H.          H.         H.         H.     =  certainly  negative. 

H.  =  complete  hemolysis;  M.  H.  =  marked  hemolysis;  S.  H.  =  slight  hemolysis; 
the  dash  ( — )  =  no  hemolysis. 

If  complete  hemolysis  has  occurred  in  all  tubes  after  an  hour's 
incubation,  the  cells  are  regarded  as  being  hypersensitive  to  cobra 
venom.  This  occurs  as  a  rule  in  primary  syphilis  and  in  active  tuber- 
culosis. 


410  VENOM   HEMOLYSIS 

PRACTICAL  VALUE  OF  THE  VENOM  TEST  IN  SYPHILIS 

1.  While  the  test  is  much  simpler  than  the  Wassermann  reaction  and 
there  is  less  possibility  for  errors  in  technic  to  creep  in,  it  possesses  but 
two  other  advantages,  namely:    (1)  It  may  react  positively  in  latent 
or  tertiary  syphilis  when  the  Wassermann  reaction  may  be  negative,  and 
(2)  it  may  react  positively  in  treated  syphilitic  cases  when  the  Wasser- 
mann reaction  is  negative,  and  thus  point  to  a  continuation  of  treatment. 
Corson- White  and  Ludlum 1  found  94  per  cent.,  Schwartz2  69.3  per  cent., 
and  Stone  and  Schottstaedt 3  90.9  per  cent,  of  positive  reactions  in  the 
active  stages  of  syphilis. 

2.  The  test  is  positive  in  but  about  20  per  cent,  of  cases  of  tabes 
dorsalis  and  general  paralysis  (White  and  Ludlum),  a  finding  obviously 
inferior  to  the  Wassermann  reaction. 

3.  During  primary  syphilis  the  cells  are  hypersensitive  and  positive 
reactions  are  but  occasionally  obtained. 

4.  Positive  reactions  may  occur  in  cancer,  but  otherwise  the  test  is 
quite  specific,  and  may,  in  selected  cases,  prove  a  valuable  adjunct  to  the 
Wassermann  reaction.     However,  with  a  more  improved  technic  in 
performing  the  Wassermann  reaction,   and  especially  if  antigens  re- 
enforced  with  cholesterin  are  used,  the  venom  test  is  inferior  to  the 
Wassermann.     In  cases  where  syphilis  or  tuberculosis  of  the  lungs  is 
to  be  differentiated,  a  negative  venom  test  would  indicate  tuberculosis, 
as  in  this  disease  the  cells  are  hypersensitive. 


THE  PSYCHO-REACTION  OF  MUCH 

Normal  serum,  when  added  to  a  lytic  dose  of  cobra  venom  and  human 
red  blood-cells,  will  not  interfere  with  hemolysis.  According  to  Much 
and  Holzman,4  however,  if  the  serum  obtained  from  a  patient  suffering 
from  depressive  mania  or  dementia  prsecox  is  added  to  the  mixture  of 
venom  and  human  red  blood-cells,  the  expected  hemolysis  does  not  take 
place. 

Technic. — A  1 :  5000  dilution  of  cobra  venom  is  prepared  by  diluting 
1  c.c.  of  the  stock  dilution  (p.  407)  with  4  c.c.  of  normal  saline  solu- 
tion. Enough  of  the  patient's  blood  is  collected  from  a  vein  at  the  elbow 
to  yield  at  least  1.5  c.c.  of  serum;  heat  the  serum  to  55°  C.  for  an  hour. 
Prepare  a  5  per  cent,  suspension  of  washed  human  blood-cells.  An  effort 

1  Jour.  Nervous  and  Mental  Diseases,  1910,  xxxvii,  721. 

2  New  York  Medical  Journal,  1912,  xcv,  23. 

3  Archiv.  of  Int.  Med.,  1912,  x,  8.  4  Munch,  med.  Wochenschr.,  1909,  20. 


THE    PSYCHO-REACTION    OF   MUCH  411 

should  be  made,  if  possible,  to  use  as  a  control  the  cells  of  a  person 
which  are  known  to  be  readily  hemolyzed  by  1  c.c.  of  the  1 :  5000  dilution 
of  venom  in  half  an  hour. 

Into  a  series  of  three  small  test-tubes  place  0.4  c.c.  of  the  patient's 
serum  and  decreasing  amounts  of  venom — 1, 0.8,  and  0.5  c.c.  respectively. 
Next  add  to  each  tube  1  c.c.  of  the  blood-corpuscle  suspension.  The 
total  volume  in  each  tube  is  brought  up  to  2.5  c.c.  by  the  addition  of 
normal  saline  solution. 

A  similar  series  of  tubes  should  be  set  up  as  controls,  the  patient's 
serum  being  omitted.  The  following  table  shows  the  arrangement: 

Tube  1:  0.4  c.c.  serum  +  1     c.c.  venom  +  1  c.c.  blood. 

Tube  2:  0.4  c.c.  serum  +  0.8  c.c.  venom  +  1  c.c.  blood. 

Tube  3:  0.4  c.c.  serum  +  0.5  c.c.  venom  +  1  c.c.  blood. 

Tube  4:  1     c.c.  venom  +  1  c.c.  blood. 

Tube  5:  0.8  c.c.  venom  +  1  c.c.  blood. 

Tube  6:  0.5  c.c.  venom  +  1  c.c.  blood. 

Tube  7:0  1  c.c.  blood. 

The  tubes  are  shaken  gently  and  incubated  for  an  hour  at  37°  C., 
when  a  preliminary  reading  is  made.  If  the  control  tubes  4,  5  and  6 
are  hemolyzed,  a  positive  reaction  would  be  indicated  by  the  inhibition 
of  hemolysis  in  the  first  three  tubes,  1,  2  and  3.  Control  tube  4  at  least 
should  be  completely  hemolyzed  at  the  end  of  half  an  hour,  and  in  a 
positive  reaction  tube  1,  containing  the  same  amount  of  venom  with  the 
patient's  serum,  will  show  inhibition  of  hemolysis.  . 

Practical  Value.— Corson.- White  and  Ludlum1  have  found  the  reac- 
tion positive  in  80  per  cent,  of  cases  of  the  catatonic  form  of  dementia 
pracox  and  in  62  per  cent,  of  the  hebephrenic  type.  Only  3  out  of  37 
cases  of  manic-depressive  insanity  reacted  positively.  Among  controls 
with  serums  of  other  diseases  positive  reactions  were  secured  in  one  case 
each  of  cerebrospinal  syphilis,  tertiary  lues,  exophthalmic  goiter,  and 
confusional  insanity,  and  in  two  cases  each  of  general  paralysis  and 
epilepsy.  The  afore-mentioned  observers  claim,  however,  that  if  the 
venom  is  carefully  standardized  with  blood-cells  that  are  completely 
hemolyzed  in  1 : 5000  dilution  of  venom  in  thirty  minutes,  the  reaction 
possesses  some  diagnostic  value,  having  been  found,  under  these  condi- 
tions, to  yield  positive  reactions  in  87  per  cent,  of  cases  of  dementia 
praecox  and  in  100  per  cent,  of  catatonics. 

According  to  Citron,  the  reaction  is  probably  due  to  interference 
with  hemolysis  by  an  increase  in  the  cholesterin  of  the  serum — a  pos- 
1  Jour.  Nerv.  and  Mental  Diseases,  1910,  xxxvii,  721. 


412  VENOM   HEMOLYSIS 

sibility  more  apt  to  occur  in  diseases  of  the  central  nervous  system  than 
in  any  physiologic  or  other  pathologic  condition. 


VENOM  HEMOLYSIS  IN  TUBERCULOSIS 

Calmette  found  that  the  blood  of  tuberculous  patients  may  activate 
cobra  hemolysin  in  very  small  doses,  and  upon  this  observation  he 
devised  a  test  that  yielded  about  65  per  cent,  of  positive  reactions  in 
tuberculosis.  Positive  reactions  have,  however,  been  found  in  other 
diseases,  and  the  practical  value  of  the  test  has  not  been  established. 


VENOM  HEMOLYSIS  IN  CANCER 

Although  the  red  blood-corpuscles  of  the  horse  may  be  hemolyzed  by 
venom  without  the  aid  of  serum,  Kraus,  Graff  and  Ranzi 1>2  found  that 
about  70  per  cent,  of  cancer  serums  considerably  hastened  and  aided 
the  hemolytic  process. 

A  1 : 5000  dilution  of  venom  is  used.  In  two  series  of  four  tubes 
each  place  respectively  0.1,  0.2,  0.3,  and  0.5  c.c.  of  the  patient's  serum 
(heated);  to  the  first  series  add  0.3  c.c.  of  the  venom  solution,  and  to  the 
second  series  0.15  c.c.  of  the  same.  To  each  of  the  tubes  in  the  series 
add  5  drops  of  a  10  per  cent,  suspension  of  washed  horse  corpuscles; 
shake  thoroughly  and  incubate  at  37°  C.  Inspect  the  tubes  at  the 
end  of  fifteen  and  thirty  minutes,  and  then  after  one,  two,  and  three 
hours. 

Positive  reactions  have  also  been  found  in  pregnancy  after  the  fourth 
month,  in  icterus,  advanced  tuberculosis,  and  other  diseases. 

1  Wien.  klin.  Wochenschr.,  1911,  No.  28. 

2  Munch,  med.  Wochenschr.,  1912,  No.  59,  574. 


CHAPTER  XXII 

PRINCIPLES  OF  THE  PHENOMENON  OF  COMPLEMENT 

FIXATION 

Historic. — With  Bordet's  discovery  of  the  hemolysins  in  1898, 
and  his  demonstration  of  the  role  of  the  antibody  or  sensitizer  and  alexin 
in  the  process,  new  light  was  thrown  upon  the  bacteriolysins,  and  the 
close  analogy  between  hemolysis  and  bacteriolysis  soon  became  apparent. 
Bordet 's  discoveries  were  quickly  verified  by  Ehrlich  and  Morgenroth 
and  the  German  school  in  general,  although  his  views  regarding  the 
mechanism  of  the  processes  were  questioned.  The  controversy  soon 
centered  upon  the  question  of  the  unity  or  the  multiplicity  of  alexins 
or  complements.  Bordet  at  this  time  advanced  his  belief  in  the  existence 
of  one  alexin  or  complement  that  would  act  with  any  sensitizer  or  ambo- 
ceptor,  and  he  still  maintains  this  view.  One  of  the  experiments  con- 
ducted by  him,  and  later  made  in  conjunction  with  his  pupil,  Gengou, 
in  support  of  his  theory,  is  now  known  as  the  Bordet-Gengou  phenomenon 
of  complement  fixation.  This  has  become  widely  known  as  the  precursor 
of  all  complement-fixation  tests,  and  is  the  basis  of  the  well-known  and 
invaluable  Wassermann  reaction  for  the  diagnosis  of  syphilis. 

In  devising  the  technic  of  this  important  method,  Bordet's  main 
object  was  to  show  that  the  complement  in  a  normal  serum  would  unite 
with  either  a  bacteriolytic  or  a  hemolytic  amboceptor,  and  that,  by 
furnishing  sufficient  of  either  amboceptor,  all  the  complement  may  be 
"  fixed."  He  argued  that  if  two  or  more  complements  existed  in  the 
same  serum,  as  was  held  by  Ehrlich,  they  would  demonstrate  their 
presence  by  exhibiting  different  affinities  for  these  widely  varying 
amboceptors. 

Prior  to  this  Bordet  had  shown  that  the  addition  of  a  small  amount  of 
normal  serum  to  an  immune  hemolysin  would  result  in  lysis  of  the  homol- 
ogous corpuscles,  and  that  the  process  could  not  take  place  without  the 
alexin.  He  then  mixed  an  emulsion  of  pest  bacilli  with  antipest  serum  and 
added  a  small  amount  of  normal,  unheated  guinea-pig  serum  to  supply 
the  alexin  or  complement.  After  allowing  the  mixture  to  stand  for  four 
hours  at  room  temperature,  it  was  sought  to  determine  whether  the 
alexin  had  been  fixed  by  pest  antigen  and  pest  amboceptor,  or  whether 

413 


414  PHENOMENON    OF    COMPLEMENT  FIXATION 

it  was  free  in  the  fluid.  Bordet  knew  that  the  normal  alexin  serum  used 
in  the  experiment  could  produce  hemolysis  of  corpuscles  with  a  homol- 
ogous amboceptor,  so  he  tested  for  free  alexin  by  subsequently  adding 
to  the  mixture  anti-rabbit  hemolysin  and  rabbit  corpuscles.  Hemolysis 
did  not  occur,  because  the  alexin  or  complement  had  been  bound  by  the 
pest  antigen  and  amboceptor.  When  a  normal  serum  was  substituted 
for  the  antipest  serum,  hemolysis  occurred,  because  the  normal  serum 
contained  no  sensitizers  or  amboceptors  that  could  unite  with  the  pest 
bacilli  and  "fix"  the  alexin,  which,  therefore,  remained  free,  and  when 
the  hemolysin  and  red  corpuscles  were  subsequently  added,  united  with 
them  to  lyse  the  red  cells.  In  this  way  the  corpuscles  and  hemolysin 
served  as  indicators  for  free  or  unfixed  alexin  or  complement,  just  as 
litmus  or  phenolphthalein  may  be  used  as  a  test  for  the  presence  of  an 
acid  or  an  alkali. 

By  showing,  in  this  manner,  that  the  complement  of  a  serum  could 
be  fixed  by  either  bacteriolytic  or  hemolytic  amboceptors,  Bordet 
endeavored  to  support  his  views  on  the  unity  of  complement.  Ehrlich 
and  Morgenroth  later  verified  his  findings,  and  in  addition,  by  more 
delicate  and  complicated  experiments,  showed  that  many  complements 
may  be  present  in  a  serum,  a  fact  manifested  by  a  different  rate  of 
absorption,  by  the  action  of  specific  anticomplements,  etc.,  as  mentioned 
in  a  previous  chapter. 

While  Ehrlich's  theory  as  to  the  multiplicity  of  complements  has 
been  widely  accepted,  the  subject  really  possesses  greater  academic 
than  practical  interest,  for  experience  has  shown  that  the  results  are  the 
same  in  complement-fixation  tests  at  least,  regardless  of  whether  we 
believe  in  the  unity  or  in  the  multiplicity  of  complement,  as  under 
ordinary  conditions  the  complement  or  complements  in  a  given  quantity 
of  serum  are  capable  of  being  absorbed  by  bacteriolytic,  hemolytic, 
or  other  amboceptors. 

Gengou  showed  later  that  not  only  cellular  antigens,  such  as  bacteria 
and  red  blood-cells,  are  capable  of  stimulating  the  production  of  ambo- 
ceptors, but  that  the  proteins  in  solution,  such  as  serum  and  milk,  may 
produce  complement-binding  amboceptors  in  addition  to  precipitins. 
This  subject  was  later  studied  more  extensively  by  Moreschi,  whose 
interest  became  aroused  as  the  result  of  his  theoretic  studies  upon 
anticomplements.  This  investigator  observed  that,  upon  mixing  a 
soluble  protein  with  its  antiserum  precipitation  occurred  and  the  existing 
complement  disappeared,  a  coincidence  that  led  him  to  assert  that  the 
complement  disappeared  because  it  was  carried  down  mechanically  in 


HISTORIC  415 

the  precipitate.  This  explanation  naturally  had  the  effect  of  leading 
many  to  assume  that  the  Bordet-Gengou  phenomenon  of  complement 
fixation  may  be  the  result  of  a  similar  precipitation  process,  and  led  to 
many  interesting  and  valuable  investigations,  especially  those  made  by 
Gay.  It  is  now  generally  agreed,  however,  that  protein  amboceptors 
are  formed,  and  that  actual  complement  fixation  occurs  independently 
of  precipitation.  Later  Neisser  and  Sachs  elaborated  on  Gengou's 
studies,  and  perfected  a  complement-fixation  technic  for  the  differen- 
tiation of  proteins  that  is  much  more  delicate  than  the  precipitin  test, 
and  serves  to  demonstrate  and  differentiate  traces  of  protein,  as  in 
blood-stains,  so  minute  in  quantity  as  not  to  be  appreciable  by  the 
precipitin  test. 

Widal  and  Lesourd  applied  the  Bordet-Gengou  reaction  to  the 
diagnosis  of  typhoid  fever,  using  an  emulsion  of  typhoid  bacilli  and  the 
serum  of  a  typhoid-fever  patient,  and  found  that  a  positive  reaction 
occurred  more  frequently  and  earlier  than  the  agglutination  test.  These 
observations  were  made  soon  after  Bordet  and  Gengou's  discovery,  and 
were  probably  the  first  direct  and  practical  application  of  a  complement- 
fixation  technic  in  diagnosis.  It  was  not  until  several  years  later, 
however,  that  the  possibilities  of  the  method  were  seriously  considered. 

Hitherto  most  experiments  were  conducted  with  known  antigens  and 
their  antibodies.  It  was  shown,  especially  in  the  work  of  Neisser  and 
Sachs  on  protein  differentiation,  that  when  an  antigen  and  its  specific 
antibody  are  present  complement  is  absorbed,  and  the  specific  relation 
existing  between  these  bodies  was  again  emphasized.  Hence  in  a  com- 
pkment-fixation  test,  if  the  antibody  is  known  the  antigen  may  be  found, 
or  if  the  antigen  is  known  the  antibody  may  be  found,  the  detection  in  either 
instance  depending  upon  whether  or  not  complement  is  absorbed,  this  being 
decided  by  adding  corpuscles  and  their  amboceptors  to  the  mixture,  the 
absence  or  the  occurrence  of  hemolysis  determining  this  point.  In  this 
manner  Neisser  and  Sachs  were  able  to  diagnose  the  nature  of  blood- 
stains by  using  solutions  of  the  suspected  stains  as  antigen,  and  adding, 
in  different  experiments,  known  antiserums  secured  by  injecting  rabbits 
with  various  bloods.  When  a  positive  complement-fixation  reaction 
occurred,  they  concluded  that  the  antigen  of  the  blood  corresponded  to 
the  known  antibody,  and  they  were  thus  able  to  identify  the  species  of 
animal  from  which  the  blood  in  the  stain  was  derived. 

Wassermann  and  Sachs,  encouraged  by  these  results,  endeavored, 
by  complement-fixation  tests,  to  show  the  existence  of  antigens  in  dis- 
eased organs,  using  tuberculous  glands  and  lungs  with  an  antituberculous 


416  PHENOMENON    OF   COMPLEMENT  FIXATION 

serum  and  the  serums  of  tuberculous  persons.  Complement  fixation 
occurred  under  certain  circumstances,  and  these  are  discussed  more 
fully  in  Chapter  XXIV.  These  investigations  finally  led  to  the  serum 
diagnosis  of  syphilis,  in  which  the  antigen  was  supplied  by  tissues 
containing  large  numbers  of  the  Spirocheta  pallida.  By  furnishing  the 
antigen  it  was  hoped  that  the  luetic  antibody  could  be  detected  in  the 
body-fluids  through  the  absorption  of  complement,  by  the  union  of  the 
antigen  and  its  antibody  in  the  complement-fixation  test.  Although 
the  primary  results  were  somewhat  discouraging,  the  possibility  was 
shown  to  exist,  and  while  the  original  theories  regarding  the  specific 
nature  of  the  antigen-antibody  reaction  have  been  modified  by  subse- 
quent discoveries,  nevertheless  this  reaction  of  Wassermann,  Neisser 
and  Bruck,  and  Detre  has  proved  itself  one  of  the  most  valuable  diag- 
nostic procedures  known. 


THE  ORIGINAL  COMPLEMENT-FIXATION  METHOD  OF  BORDET 

In  a  paper  published  in  1901  Bordet 1  gives  the  results  attained  with 
three  different  antigens  and  their  respective  antiserums — pest,  typhoid, 
and  Proteus  vulgaris.  The  details  of  the  technic  employed  with  an 
antigen  of  pest  bacilli  and  an  antipest  horse  serum  are  given. 

(a)  The  antigen  consisted  of  twenty-four-hour  cultures  of  pest  bacilli 
in  normal  salt  solution,  making  a  somewhat  concentrated  emulsion. 

(6)  The  antipest  horse  serum  was  heated  for  half  an  hour  at  56°  C., 
to  remove  the  alexin  or  complement.  Normal  horse  serum  (heated) 
was  also  employed  as  a  negative  control. 

(c)  As  alexin,  the  fresh  serum  of  a  normal  guinea-pig  was  used. 

(d)  The  substance  sensibilisatrice  or  hemolysin  consisted  of  the  inac- 
tivated serum  of  a  guinea-pig  injected  with  rabbit's  red  blood-cells. 

(e)  The  corpuscles  of  the  rabbit  were  washed,  to  free  them  of  alexin, 
and  were  used  as  the  indicatory  antigen. 

To  0.4  c.c.  of  the  pest  emulsion  1.2  c.c.  of  inactivated  antipest 
serum  and  0.2.  c.c.  of  guinea-pig  alexin  were  added.  This  mixture  was 
allowed  to  remain  at  the  ordinary  laboratory  temperature  (18°-20°  C.) 
for  several  hours.  In  order  to  ascertain  whether  or  not  the  alexin  had 
been  absorbed,  hemolysin  and  erythrocytes  were  added  to  the  mixture. 
This  was  accomplished  by  sensitizing  about  20  drops  of  washed  rabbit's 
cells  with  2  c.c.  of  inactivated  hemolysin  for  about  fifteen  minutes,  and 
adding  two  drops  to  each  of  the  test-tubes.  Hemolysis  did  not  occur 
1  Bordet:  Ann.  de  1'Inst.  Pasteur,  1901,  xv,  19. 


ORIGINAL  COMPLEMENT-FIXATION   METHOD   OF   BORDET      417 


because  the  alexin  had  been  fixed  by  the  pest  antigen  and  antibody.  A 
similar  test,  conducted  with  normal  serum,  hemolyzed  in  a  few  minutes 
because  the  complement  or  alexin  remained  free  in  the  mixture.  Even 
at  this  early  stage  Bordet  included  the  important  controls  on  his  antigen 
and  serums  that  are  so  necessary  in  all  complement-fixation  tests. 

The  following  table  gives  the  original  details  and  results  of  the  first 
complement-fixation  experiment  with  pest  antigens  and  antipest  serum: 

TABLE   12.— THE   ORIGINAL  BORDET-GENGOU  COMPLEMENT- 
FIXATION  REACTION 


TUBE 

ANTIGEN, 
C.c. 

SERUM 

ALEXIN, 
C.c. 

HEMOLYSIN  AND 
ERYTHROCYTES 

RESULTS 

(a)  

0.4 

1.2  c.c.  inactivated 

0.2 

2  drops  sensitized 

No  hemol- 

(b)  
(c)  

(d) 

0.4 

antipest  serum 
1  2  c.c.  inactivated 
normal  serum 
1.2  c.c.  inactivated 
antipest  serum 
1  2  c  c  inactivated 

0.2 
0.2 
0.2 

rabbit's  blood 
2  drops  sensitized 
rabbit's  blood 
2  drops  sensitized 
rabbit's  blood 
2  drops  sensitized 

ysis 
Complete 
hemolysis 
Complete 
hemolysis 
Complete 

(e)  
(/)  

0.4 
0.4 

normal  serum 
1.2  c.c.  inactivated 
antipest  serum 
1.2  c.c.  inactivated 
normal  serum 

rabbit's  blood 
2  drops  sensitized 
rabbit's  blood 
2  drops  sensitized 
rabbit's  blood 

hemolysis 
No  hemol- 
ysis 
No  hemol- 
ysis 

Mechanism  of  Complement  Fixation. — The  divergent  views  of 
Bordet  and  Ehrlich  on  the  mechanism  of  antigen-amboceptor  action 
have  been  given  elsewhere.  Bordet  believes  that  the  antibody  unites 
directly  with  the  antigen,  and  serves  to  sensitize  and  prepare  it  for  direct 
union  with  the  alexin  or  complement,  in  a  manner  similar  to  that  of 
using  a  mordant  in  aiding  the  penetration  of  a  dye-stuff.  In  the  absence 
of  the  homologous  and  specific  antibody  (sensitizer),  the  antigen  is 
incapable  of  absorbing  more  than  very  small  amounts  of  complement  or 
none  at  all.  In  the  absence  of  antigen,  the  sensitizer  and  complement  do 
not  unite,  or  unite  to  but  a  very  slight  degree.  The  important  require-*' 
ment  for  complement  fixation  is,  therefore,  an  antigen  that  has  been 
sensitized  by  the  antibody,  and  in  this  manner  has  an  increased  combin- 
ing affinity  for  complement. 

According  to  Ehrlich  and  Morgenroth,  however,  the  complement  does 
not  unite  directly  with  the  antigen,  but  only  indirectly  through  the 
antibody,  which  acts  as  a  connecting  link  or  amboceptor  between  antigen 
and  complement.  Antigen  alone,  or  even  amboceptor  alone,  binds  the 
complement  only  very  slightly  or  not  at  all.  The  important  require- 
ment for  complement  fixation  is  an  amboceptor  attached  to  its  homol- 
27 


418  PHENOMENON    OF   COMPLEMENT  FIXATION 

ogous  or  suitable  antigen,  which  increases  the  affinity  and  fixing  power  of 
the  amboceptor  for  the  complement.  (See  Fig.  85) . 

Complement-fixation  tests  also  serve  to  demonstrate  that  absorption 
of  complement  is  not  necessarily  followed  by  lysis  of  the  antigen.  For 
example,  anthrax  and  pest  bacilli,  when  mixed  with  their  homologous 
amboceptors  and  complement,  show  no  bacteriolysis  or  but  a  very 
slight  reaction.  The  erroneous  conclusion  thus  reached,  that  these 
serums  contained  no  amboceptors,  was  disproved  by  Bordet,  who  demon- 
strated that  they  contained  amboceptors  and  that  complement  was 
absorbed  or  fixed  although  bacteriolysis  had  not  taken  place.  Whether 
the  difference  here  depends  upon  variations  in  the  nature  pf  lytic  and 
non-lytic  amboceptors  or  whether  it  is  due  to  the  relative  amounts  of  an 
amboceptor  or  the  construction  and  constitution  of  the  antigen  is  not 
known.  It  would  appear,  however,  that  the  last  two  possibilities  are 
largely  concerned,  although,  so  far  as  complement  fixation  is  concerned, 
it  is  immaterial  whether  or  not  bacteriolysis  occurs. 

Complement-fixing  antibodies,  therefore,  are  probably  all  in  the 
nature  of  amboceptors,  and  these  are  to  be  found  in  varying  amounts  in 
practically  all  immune  serums,  including  antitoxic,  agglutinating,  and 
precipitating  serums. 


NON-SPECIFIC  COMPLEMENT  FIXATION 

The  importance  of  having  proper  controls  in  practically  all  tests  is 
especially  to  be  emphasized  in  complement-fixation  work.  While  the 
underlying  principles  are  readily  understood  and  the  technic  is  compar- 
atively simple,  there  are,  however,  many  sources  of  error  that  require  a 
thorough  understanding  in  order  that  an  intelligent  and  reliable  com- 
plement-fixation reaction  may  be  secured.  These  refer  mainly  to  non- 
specific fixation  of  complement  and  to  quantitative  factors  governing 
complement-fixation  technic. 

Formed  elements,  such  as  bacteria  and  tissue-cells,  as  well  as  various  ' 
organic  and  inorganic  material,  may  fix  complement  by  themselves,  i.  e., 
in  a  non-specific  manner,  and  chemicals,  such  as  acids  and  alkalis,  may 
destroy  it. 

1.  An  antigen  alone  in  certain  amounts  may  absorb  complement.    ThisV 
anticomplementary  dose  of  an  antigen,  as  it  is  called,  must  be  determined 
beforehand  by  a  process  of  titration  when  increasing  amounts  of  antigen 
are  mixed  with  a  constant  dose  of  complement,  hemolysin,  and  corpuscles 
and  the  anticomplementary  action  of  the  antigen  noted  by  the  results 


NON-SPECIFIC    COMPLEMENT   FIXATION  419 

of  hemolysis,  i.  e.,  if  a  small  amount  of  complement  is  absorbed,  hemol- 
ysis  will  be  correspondingly  incomplete,  if  all  complement  is  absorbed, 
hemolysis  does  not  occur  at  all.  If,  therefore,  in  any  complement-fixa- 
tion test  the  antigen  is  used  in  an  amount  that  will  give  this  non-specific 
absorption,  even  to  a  slight  degree,  a  grave  source  of  error  is  introduced. 

As  a  general  rule  for  all  complement-fixation  tests,  the  dose  of  antigen 
employed  should  never  be  more  than  one-fourth  or  one-half  of  its 
anticomplementary  dose  (that  amount  which  of  itself  is  capable  of 
non-specific  complement-fixation). 

2.  A  serum  may  of  itself  absorb  a  small  amount  of  complement,  espe- 
cially if  it  is  old  or  infected  with  bacteria.  This  is  known  popularly  as 
the  anticomplementary  action  of  a  serum,  and  in  every  complement- 
fixation  test  in  order  to  detect  this  condition  a  proper  control,  consisting 
of  the  dose  of  serum  used  plus  complement,  hemolysin,  and  corpuscles 
is  required,  the  non-specific  absorption  of  complement  being  determined 
by  the  results  of  hemolysis. 

Moreover,  perfectly  fresh  serums  may  show  this  non-specific  absorp- 
tion of  complement  to  a  slight  degree,  especially  in  the  presence  of  the 
lipoidal  extracts  used  as  "antigens"  in  the  Wassermann  reaction. 

Heating  a  serum  to  56°  C.  for  half  an  hour  largely  removes  this 
anticomplementary  effect  of  serums,  unless  they  are  quite  old  and  in- 
fected; accordingly,  heated  serums  are  used  almost  exclusively  in  com- 
plement-fixation tests.  This  is  usually  called  inactivation,  or  the  removal 
of  native  complement  from  a  serum,  but  in  the  majority  of  instances 
the  complement  of  a  serum  generally  deteriorates  rapidly,  and  the  serum 
is  heated  mainly  for  the  purpose  of  removing  its  anticomplementary 
action,  i.  e.,  its  ability  to  effect  non-specific  absorption  of  complement. 

By  referring  to  the  original  Bordet  experiment,  it  will  be  observed 
that  this  investigator  controlled  any  non-specific  absorption  of  comple- 
ment by  both  the  immune  and  the  normal  serum  in  tubes  C  and  D  of 
the  series  by  using  the  full  dose  of  these  serums,  with  a  similar  amount  of 
complement,  and  noting  that  hemolysis  was  complete.  His  controls, 
E  and  F,  were  to  determine  if  the  process  of  inactivation  or  removal  of 
native  complement  from  the  two  serums  was  complete,  and  the  total  ab- 
sence of  hemolysis  showed  that  it  was.  His  control  on  the  anticomple- 
mentary action  of  the  antigen  was  also  included  in  tube  D,  for  if  the 
emulsion  alone  had  absorbed  complement  to  any  degree,  hemolysis 
would  have  been  incomplete. 

Non-specific  Complement  Fixation  by  Normal  Rabbit,  Dog,  and  Mule 
Sera. — The  fresh  normal  sera  of  several  species  of  animals  may  absorb  or 


420  PHENOMENON    OF    COMPLEMENT    FIXATION 

fix  complement  with  various  lipoidal  and  bacterial  antigens  in  a  non-specific 
manner.  Schilling  and  Hoesslin1  were  probably  among  the  first  investi- 
gators to  note  this  phenomenon  with  normal  rabbit  serum.  Manteufel 
and  Woithe2  and  Browning  and  McKenzie3  have  also  noted  the  phenom- 
enon during  studies  in  experimental  trypanosomiasis.  Dohi4  examined 
the  sera  of  74  normal  rabbits,  using  as  antigen  an  alcoholic  extract  of 
syphilitic  liver,  and  found  that  39  reacted  positively  and  35  negatively. 
He  also  observed  that  heated  serum  was  more  likely  to  show  this  non- 
specific absorption  of  complement  than  unheated  serum.  Browning  and 
McKenzie,  however,  state  that  they  have  not  observed  this 'phenomenon 
when  using  serum  in  a  fresh,  or  active,  condition.  Blumenthal5  has  like- 
wise observed  positive  reactions  with  normal  rabbit  serum,  using  an 
alcoholic  extract  of  syphilitic  liver  as  antigen;  Craig  and  Nichols,6  on  the 
other  hand,  have  reported  uniformly  negative  results  with  an  alcoholic 
extract  of  syphilitic  liver.  Similar  observations  on  this  power  of  normal 
rabbit  serum  to  absorb  complement  in  the  presence  of  lipoidal  antigens 
have  been  made  by  Emmanuel7  and  by  Epstein  and  Pribram,8  the  former 
stating  that  the  administration  of  salvarsan  removes  this  property 
temporarily,  and  the  latter  making  similar  claims  for  the  mercurials. 
Casselman  and  I9  observed  a  large  percentage  of  positive  reactions  with 
the  sera  of  117  normal  rabbits  and  various  lipoidal  extracts  used  as 
antigens  in  the  Wassermann  reaction.  In  a  further  study  by  Miss  Trist 
and  I10  we  found  that  while  heated  rabbit  serum  yielded  positive  re- 
actions with  38  to  49  per  cent,  of  sera  with  lipoidal  antigens,  active  or 
unheated  sera  yielded  but  5-15  per  cent,  positive  reactions.  Bacterial 
antigens  yielded  even  higher  percentages  of  positive  reactions.  Pearce 
and  I11  have  also  found  that  the  sera  of  animals  reacting  positively  or 
negatively  generally  continue  to  react  in  the  same  manner  over  long  peri- 
ods of  observation.  Rossi,12  Miss  Trist  and  I13  have  also  found  that  the 
sera  of  a  large  percentage  of  normal  dogs  tends  to  yield  non-specific  fixa- 

1  Deutsch.  med.  Wchnschr.,  1908,  34,  1422. 

2  Centralbl.  f.  Bakteriol.,  R.,  1909,  43,  359. 

3  Jour.  Path,  and  Bacteriol.,  1909-11,  15,  182. 

4  Beitr.  z.  path.  u.  Therap.  der  Syph.,  1911,  514. 
5Berl.  klin.  Wchnschr.,  1911,  48,  1462. 

e  Jour.  Exper.  Med.,  1911,  14,  206. 
*Berl.  klin.  Wchnschr.,  1911,  48,  2335. 

8  Ztschr.  f.  exper.  Path.  u.  Therap.,  1909,  7,  549. 

9  Jour.  Med.  Research,  1913,  28,  369. 

»  Jour.  Infect.  Dis.,  1916,  18,  20.  u  Jour.  Infect.  Dis.,  1916,  18,  32. 

12  Ztschr.  f.  Immunitatsf.,  R.,  1909,  1,  429. 

13  Jour.  Infect.  Dis.,  1916,  18,  27. 


NON-SPECIFIC    COMPLEMENT    FIXATION  421 

tion  with  various  lipoidal  and  bacterial  antigens.  The  mechanism  of  this 
phenomenon  is  not  understood;  it  would  appear  that  the  complement- 
absorbing  body  is  in  both  the  serum  lipoids  and  proteins.1  Heating 
these  sera  at  56°  C.  for  half  an  hour  greatly  increases  their  property  for 
non-specific  fixation  of  complement;  heating  at  62°  C.  for  the  same  period 
lessens  the  tendency.2  The  subject  is  of  great  importance  in  view  of 
the  frequency  with  which  complement-fixation  studies  are  conducted 
with  rabbit,  dog,  and  mule  sera. 

Quantitative  Factors  in  Complement-fixation  Tests. — From  what 
has  been  said  it  will,  therefore,  readily  be  appreciated  that  complement- 
fixation  tests  are  largely  quantitative.  Equally  fallacious  results  may 
be  obtained  by  using  too  large  or  too  small  amounts  of  the  various 
ingredients. 

While  it  is  possible  to  use  too  large  quantities  of  antigen,  so  that 
non-specific  absorption  of  complement  occurs,  leading  to  false  positive 
reactions,  it  is  also  possible  to  use  an  amount  so  small  that  any  specific 
absorption  of  complement  by  antigen  and  antibody  cannot  readily  be 
detected. 

The  same  is  true,  but  to  a  much  less  extent,  of  the  immune  serum, 
for  while  too  large  amounts  of  serum  may  lead  to  non-specific  fixation  of 
complement,  surprisingly  small  amounts  may  give  well-marked  specific 
fixation,  this  factor  depending,  of  course,  upon  the  quantity  of  anti- 
bodies contained  in  the  serum. 

Of  even  greater  importance  are  the  quantity  of  complement  em- 
ployed and  the  proper  adjustment  of  the  hemolytic  system,  composed  of 
complement,  hemolysin,  and  corpuscles. 

Too  large  an  amount  of  complement  may  furnish  sufficient  to  sat- 
isfy the  amboceptors  of  an  immune  serum  united  with  the  antigen, 
with  enough  free  complement  left  over  to  produce  partial  or  complete 
hemolysis  when  corpuscles  and  hemolysin  are  subsequently  added.  In 
this  manner  specific  complement  fixation  would  be  overlooked  and  a 
false  negative  reaction  secured. 

It  is  also  possible  to  use  too  small  an  amount  of  complement,  with 
relatively  large  doses  of  serum  and  antigen,  so  that  the  complement 
becomes  unduly  susceptible  to  non-specific  fixation  and  consequently 
false  positive  reactions  may  be  secured. 

It  has  previously  been  explained  that  an  excess  of  hemolysin  may 
offset  any  slight  deficiency  in  the  amount  of  complement.  For  instance, 
if  a  small  amount  of  complement  is  specifically  fixed  by  an  antigen  and 

1  Jour.  Infect.  Dis.,  1916,  18,  46.  2  Jour.  Infect.  Dis.,  1916,  18,  64. 


422  PHENOMENON    OF    COMPLEMENT    FIXATION 

its  amboceptor,  the  addition  of  too  large  an  amount  of  hemolysin  may 
result  in  complete  hemolysis  of  the  corpuscle,  and  thus  overshadow  the 
slight  but  specific  fixation  of  complement. 

On  the  other  hand,  hemolysis  cannot  be  complete  if  the  dose  of 
amboceptor  is  too  small.  With  a  given  dose  of  corpuscles  and  comple- 
ment a  certain  amount  of  hemolysin  is  necessary  to  produce  hemolysis, 
this  dose  being  determined  by  a  process  of  titration,  as  described  in  a 
previous  chapter.  If  less  than  this  dose  is  used,  but  the  amounts  of 
corpuscles  and  complement  remain  the  same,  hemolysis  will  be  corres- 
pondingly incomplete  and  lead  to  false  positive  reactions. 

A  very  important  feature  of  all  complement-fixation  tests  will  be 
seen  to  be  a  proper  and  accurate  adjustment  of  the  hemolytic  system. 
Taking  arbitrary  amounts  of  corpuscles  and  hemolysin  as  constants, 
the  quantity  of  complement  necessary  to  produce  hemolysis  may  be 
determined  (titration  of  complement);  or,  taking  corpuscles  and  com- 
plement as  constants,  the  amount  of  hemolysin  necessary  to  effect 
complete  hemolysis  may  be  determined.  One  or  the  other  or  both  titra- 
tions  should  be  made  before  the  main  test  is  attempted,  in  order  to 
avoid  using  an  excess  or  too  little  of  either  ingredient.  If  the  exact  unit 
of  complement  and  hemolysin  are  used,  the  results  must  be  very  care- 
fully guarded,  because  in  a  general  way  all  antigens  and  serums  exert  a 
slight  anticomplementary  action  that  may  yield  results  that  will  be 
interpreted  as  weak  positive  reactions.  For  this  reason  the  original 
complement-fixation  tests  invariably  called  for  a  slight  excess  of  com- 
plement or  hemolysin  or  both,  to  allow  for  possible  non-specific  com- 
plement fixation,  and  this  is  a  good  general  rule  that  makes  the  reaction 
somewhat  less  delicate,  but  more  reliable  in  the  long  run,  especially  for 
inexperienced  workers. 

Complements  of  different  species  of  animals  act  differently  in  activat- 
ing a  hemolytic  amboceptor  and  toward  fixation  by  antigen-antibody  com- 
binations. For  instance,  a  complement  from  one  animal  may  readily 
enough  combine  with  a  hemolytic  amboceptor  to  produce  hemolysis, 
but  will  not  lend  itself  for  fixation,  and  is,  therefore,  unfit  for  comple- 
ment-fixation tests.  Noguchi  and  Bronfenbrenner  have  found  guinea- 
pig  serum  most  suitable  from  all  standpoints,  but  it  is  important  to 
remember  that  the  complementary  activity  of  the  serums  from  different 
guinea-pigs  varies,  and,  therefore,  it  is  necessary  to  titrate  each  comple- 
ment serum  or  hemolysin,  i.  e.,  adjust  the  hemolytic  system,  before  the 
main  test  is  conducted. 

These  quantitative  factors  are  of  great  importance,  and  complicate 


PRACTICAL    APPLICATIONS  423 

any  complement-fixation  method,  but  efforts  to  circumvent  or  ignore 
them  are  likely  to  lead  to  errors  in  technic.  A  proper  understanding  and 
appreciation  of  these  factors  constitutes  the  basis  for  reliable  work, 
whereas  less  essential  details  may  be  altered  to  conform  to  the  ideas  and 
convenience  of  the  individual  worker. 


PRACTICAL  APPLICATIONS 

It  will  be  understood,  therefore,  that  complement-fixation  reactions 
may  serve  two  primary  purposes: 

1.  With  a  known  antigen,  the  antibody  may  be  found.    This  is  the 
usual  order  in  diagnostic  tests.    For  example,  in  the  reaction  for  syphilis 
the  antigen  is  furnished  and  the  antibody  sought  for  in  the  body-fluids. 
So  specific  has  this  test  proved  in  the  diagnosis  of  this  disease  that  a 
positive  reaction  secured  with  a  proper  technic  is  regarded  as  strong 
evidence  of  the  existence  of  lues,  even  though  the  primary  lesion  had 
occurred  years  before  and  the  person  is  at  the  time  in  apparent  good 
health.    In  the  gonococcus  fixation  test  and  other  tests  of  a  similar  nature 
the  antigen  is  known  and  is  furnished,  and  the  antibody  is  tested  for  in 
the  serum. 

2.  With  a  known  antibody  the  corresponding  antigen  may  be  found. 
This  order  of  events  has  less  practical  application,  and  is  used  principally 
in  the  diagnosis  of  blood-stains  and  in  the  differentiation  of  proteins  in 
general.    It  is  also  used  in  making  special  bacteriologic  investigations, 
when  an  organism  may  be  identified  by  specific  complement  fixation 
with  its  known  antibody  serum.    In  these  instances  the  antibody  serum 
is  secured  by  immunizing  rabbits  with  a  known  antigen,  the  immune 
serum  then  being  used  for  selecting  the  antigen  in  unknown  substances 
and  mixtures. 

Complement-fixation  methods  have  their  greatest  value,  and  are 
probably  best  known,  in  the  serum  diagnosis  of  syphilis — the  biologic 
syphilitic  reaction  of  Wassermann,  Neisser  and  Bruck,  and  Detre. 
Although  originally  believed  to  be  a  direct  application  of  the  specific 
Bordet-Gengou  phenomenon  of  complement  fixation,  subsequent  inves- 
tigations have  shown  that  the  antigen  need  not  be  specific,  in  the  sense 
of  containing  the  Spirocheta  pallida,  but  that  lipoidal  substances  in 
general  may  serve  as  "antigen,"  the  peculiar  and  specific  character  of 
the  reaction  depending  upon  the  nature  of  the  antibody,  which  has  a 
strong  affinity  for  lipoids,  and  in  such  a  mixture  is  capable  of  absorbing 
or  fixing  a  considerable  amount  of  complement. 


424  PHENOMENON    OF    COMPLEMENT    FIXATION 

In  no  other  disease  has  the  method  been  so  widely  employed  as  in 
syphilis,  although  it  possesses  value  in  the  serum  diagnosis  of  various 
bacterial  infections,  such  as  gonorrhea,  glanders,  typhoid  fever,  echino- 
coccus  disease,  etc.,  and  in  the  diagnosis  of  blood-stains  and  in  the 
differentiation  of  proteins  in  general. 

In  the  following  chapter  the  Wassermann  syphilitic  reaction  will  be 
considered  in  some  detail,  as  a  thorough  working  knowledge  of  this  test 
is  of  great  value,  and  serves  as  the  foundation  of  complement-fixation 
technic  in  general. 


CHAPTER  XXIII 
THE  TECHNIC  OF  COMPLEMENT-FIXATION  REACTIONS 

THE  WASSERMANN  REACTION  IN  SYPHILIS 

Historic. — Following  Bordet's  important  discovery  of  complement 
fixation  no  practical  applications  were  made  for  several  years  until 
Neisser  and  Sachs  continued  Gengou's  studies  on  protein  antigens 
and  amboceptors,  and  advocated  complement  fixation  as  a  fine  and 
delicate  method  of  control  on  the  precipitin  test  in  the  detection  and 
differentiation  of  minute  traces  of  proteins,  as  in  the  recognition  and 
diagnosis  of  blood-stains. 

Encouraged  by  these  results,  Wassermann  used  the  method  in  an 
attempt  to  discover  in  the  blood-serum,  during  the  course  of  an  infection, 
the  bacterial  proteins  derived  from  a  microorganism.  Practical  appli- 
cation proved,  however,  that  enough  of  these  proteins  did  not  exist  free 
in  the  blood  to  give  definite  complement  fixation. 

In  1905  Wassermann  and  Bruck  found  that  bacterial  extracts  may 
be  substituted  for  emulsions  of  bacteria  as  antigen  in  performing  the 
Bordet-Gengou  test,  and  that  extracts  of  diseased  organs  containing 
large  numbers  of  bacteria  or  their  products  may  be  employed.  Accord- 
ingly, these  observers  prepared  aqueous  extracts  of  tuberculous  lungs 
and  glands  and  used  them  as  antigens  in  the  study  of  complement  fixa- 
tion in  tuberculosis.  Positive  reactions  were  secured  with  an  anti- 
tuberculous  serum  and  with  the  serums  of  persons  who  had  received 
injections  of  tuberculin. 

At  this  time  Schaudinn  and  Hofmann  discovered  the  spirochete  of 
syphilis,  a  finding  that  served  to  focus  the  attention  of  the  medical  world 
upon  this  disease.  In  cooperation  with  Neisser,  who  was  conducting 
extensive  researches  on  experimental  syphilis  in  monkeys,  Wassermann 
and  Bruck  applied  the  complement-fixation  method  to  the  study  OP 
these  experimental  infections,  and  published  a  report  of  their  work  on 
May  10,  1906. 

At  first  monkeys  were  immunized  with  aqueous  extracts  of  human 
chancre,  condylomata,  syphilitic  placenta,  etc.,  and  their  serums, 
mixed  in  vitro  with  these  extracts,  were  found  to  give  the  complement- 

425 


426        THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

fixation  reaction.  Since  these  results  may  have  been  due  to  protein 
amboceptors  or  precipitins  produced  simultaneously  by  the  injection  of 
human  serum  contained  in  the  extracts,  the  experiment  was  carried  out 
with  extracts  of  bone-marrow  and  other  organs  of  syphilitic  monkeys 
used  to  obviate  this  error.  It  was  found,  however,  that  the  inactivated 
serums  of  syphilitic  monkeys  reacted  positively  with  antigens  of  either 
human  or  monkey  lesions,  and  regardless  of  whether  the  monkeys  had 
been  injected  with  human  extracts,  since,  after  ordinary  cutaneous 
infection,  their  serum  would  show  complement  fixation.  These  early 
reports  also  showed  a  high  specificity  for  complement  fixation,  as  monkey 
immune  serum  did  not  react  with  extracts  of  normal  organs  or  normal 
monkey  serum  with  extracts  of  syphilitic  organs. 

Just  fourteen  days  after  Wassermann,  Neisser,  and  Bruck  published 
their  report,  a  second  paper  on  the  same  subject  appeared,  showing  the  work 
of  Detre.  Using  aqueous  extracts  of  luetic  papules,  liver,  pancreas,  and 
tonsillar  exudate  as  antigens,  Detre  performed  the  complement-fixation 
method  with  the  serums  of  six  syphilitic  and  four  normal  persons,  finding 
positive  reactions  with  two  of  the  six  luetic  serums. 

In  1906  Wassermann  and  Plaut  studied  the  cerebrospinal  fluids  of 
41  cases  of  paresis,  and  found  positive  reactions  in  32,  4  cases  reacting 
doubtfully  and  5  negatively.  In  the  following  year  Levaditi  and  Marie 
and  Schutze  observed  positive  reactions  with  the  cerebrospinal  fluid  of 
tabetics,  whereas  Morgenroth  and  Stertz  confirmed  the  previous  finding 
in  paresis.  Since  then  numerous  investigators  have  corroborated  these 
observations,  and  while  all  the  evidence  tended  to  strengthen  the  belief 
in  the  luetic  origin  of  general  paralysis  and  tabes,  decisive  confirmation 
was  lacking  until  Noguchi  and  Moore,  in  1913,  demonstrated  the  pres- 
ence of  the  Treponema  pallidum  in  sections  of  the  cerebral  cortex. 

In  1906  Wassermann,  Neisser,  Bruck,  and  Schucht  applied  the 
complement-fixation  test  to  a  large  number  of  cases  of  syphilis  in  Neisser's 
clinic.  Aqueous  extracts  of  luetic  liver,  placenta,  glands,  chancres,  and 
gummata  were  used  as  antigens.  Of  257  cases  in  all  stages  of  the  disease, 
only  49  reacted  positively.  With  but  19  per  cent,  positive  reactions, 
the  method  did  not  appear  to  have  a  promising  future,  although  at  the 
present  time,  with  a  better  understanding  of  the  technic  and  of  the 
importance  of  quantitative  factors  that  greatly  influence  the  results, 
the  value  of  the  test  has  been  greatly  enhanced. 

As  it  appeared  that,  after  all,  no  method  of  diagnosis  was  to  be 
secured  as  the  result  of  the  demonstration  of  the  syphilitic  antibody  in 
the  body-fluids,  Neisser  and  Bruck  determined  to  return  to  earlier 


THE    WASSERMANN   REACTION    IN   SYPHILIS  427 

methods  and  attempt  to  discover  if  luetic  antigen  could  be  demonstrated 
in  the  serums  of  luetics  through  complement  fixation.  Antigens  pre- 
pared of  the  red  corpuscles  of  syphilitic  persons  gave  positive  reactions 
with  the  serums  of  highly  immunized  monkeys.  Of  160  luetic  patients, 
in  70  per  cent,  either  antigen  or  antibody  was  found.  Later,  however, 
Citron  showed  that  the  extracts  of  corpuscles  of  normal  persons  yielded 
similar  results,  which,  in  the  light  of  subsequent  discoveries,  was  due  to 
their  content  in  lipoidal  substances. 

Up  to  this  time  the  syphilitic  reaction  was  considered  as  but  a  simple 
and  direct  application  of  Bordet's  phenomenon,  requiring  a  specific 
syphilitic  antigen  before  complement  could  be  fixed  with  the  syphilis 
antibody.  In  January,  1907,  Weygandt  reported  that  he  had  obtained  a 
positive  reaction  in  tabes  with  an  extract  of  normal  spleen.  Marie 
and  Levaditi,  using  an  aqueous  extract  of  normal  fetal  liver,  secured 
positive  reactions  with  the  cerebrospinal  fluid  of  paretics,  but  observed 
that  it  was  necessary  to  use  larger  doses  than  when  extracts  of  syphilitic 
organs  were  used.  Subsequently  other  investigators,  as,  for  example; 
Fleischmann,  Michaelis,  Landsteiner,  and  Plaut,  found  that  watery 
extracts  of  normal  organs  served  to  fix  the  complement  with  luetic 
antibody.  Finally,  in  December,  1907,  a  profound  impression  was  cre- 
ated by  the  discovery  made  by  Landsteiner,  Mtiller,  and  Potzl,  that  an 
alcoholic  extract  of  guinea-pig  heart  yielded  results  equal  to  those 
obtained  with  an  aqueous  extract  of  syphilitic  liver.  These  results 
indicated  that  the  antigenic  principle  was  soluble  in  alcohol,  and  a  pro- 
longed series  of  investigations  on  the  various  lipoids  and  their  relation 
to  the  reaction  was  begun.  These  included  the  employment  of  leci- 
thin by  Forges  and  Meier;  sodium  taurocholate  and  glycocholate  by 
Levaditi  and  Yamonouchi;  cholesterin  and  vaselin  by  Fleischmann; 
oleic  acid  by  Sachs  and  Altman;  acetone-insoluble  fractions  of  alcoholic 
extracts  by  Noguchi;  and  many  other  combinations  of  various  lipoidal 
substances  by  different  investigators. 

These  dealt  a  blow  to  the  theory  of  the  Wassermann  reaction,  which, 
taken  in  conjunction  with  the  wide-spread  use  of  the  test  by  inexperi- 
enced and  unskilful  persons  and  the  many  sources  of  error,  tended  to 
retard  an  earlier  appreciation  of  the  great  value  of  the  test,  and  served  to 
swing  the  pendulum  of  medical  opinion  so  far  in  the  wrong  direction  that 
it  is  only  now,  with  a  better  understanding  of  its  possibilities  and  limita- 
tions, that  the  method  is  being  established  in  its  proper  sphere.  Posi- 
tive reactions  were  said  to  have  occurred  in  frambesia,  leprosy,  malaria, 
pellagra,  pneumonia,  scarlet  fever,  typhoid  fever,  malignant  tumors,  and 


428        THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

practically  every  other  disease  liable  to  afflict  humanity.  The  careful 
work  of  Citron  was  largely  instrumental  in  preserving  the  importance 
of  the  reaction  until  a  better  understanding  of  the  technic  resulted  in 
improved  and  more  careful  work,  with  a  greater  respect  for  the  real  value 
of  the  Wassermann  reaction. 

Although  the  true  explanation  of  the  mechanism  of  complement 
fixation  in  syphilis  is  still  lacking,  sufficient  work  has  been  done  to  show 
that  the  specific  nature  of  the  reaction  is  dependent  upon  a  peculiar 
luetic  antibody,  and  that  the  older  belief  in  the  specificity  of  antigen,  in 
so  far  as  it  insisted  upon  the  presence  of  the  Treponema  pallidum  in  the 
tissues  extracted  for  "antigen,"  is  largely  disproved.  While  the  method 
cannot  be  said  to  be  absolutely  diagnostic  of  syphilis,  since  positive 
reactions  were  had  in  frambesia  (yaws)  and  leprosy,  yet  it  is  practically 
so,  especially  in  those  countries  where  these  two  infections  are  unknown 
or  are  relatively  infrequent. 

Principles  and  Theories  of  the  Syphilitic  Reaction.  —  The  discovery 
that  the  antigen  in  the  Wassermann  reaction  is  not  necessarily  biologically 
specific,  but  may  be  furnished  by  a  variety  of  different  lipoids1  from 
normal  or  syphilitic  tissues,  opened  up  an  entirely  new  phase  of  the 
well-known  theory  of  complement  fixation,  and  separated  the  syphilitic 
reaction  from  the  classic  Bordet-Gengou  phenomenon,  as  based  upon  the 
absorption  of  fixation  of  complement  by  a  specific  antigen  and  its  anti- 
body. 

As  is  now  well  known,  the  lipoids  have  always  been  chiefly  concerned 
in  the  reaction,  although  Wassermann  and  Detre  and  their  coworkers 
naturally  ascribed  the  complement-fixing  powers  of  their  extracts  to  the 
presence  of  the  Treponema  pallidum.  It  is,  indeed,  fortunate  that  pure 
cultures  of  the  treponema  were  not  available  at  the  time  the  original 
studies  were  made,  for  these  would  naturally  have  been  employed  as 
antigen,  and  as  subsequent  work  with  pallidum  antigens  has  shown 
complement  fixation  to  be  quite  irregular  and  less  reliable  than  when 
lipoidal  extracts  are  used,  this  result,  coupled  with  the  imperfect  under- 
standing and  faulty  technic  of  the  earlier  investigations,  would  probably 
have  yielded  results  so  discouraging  as  to  constitute  weighty  drawbacks 
to  the  full  development  of  the  reaction. 

Notwithstanding  the  large  amount  of  work  that  has  been  done  in  an 
effort  to  ascertain  the  true  nature  of  the  syphilitic  reaction,  a  correct 


term  "lipoid"  ("fat-like")  is  applied  to  compounds  that  are  soluble  in 
ether,  alcohol,  chloroform,  and  benzol,  but  every  lipoid  is  not  soluble  in  all  these 
reagents. 


THE   WASSERMANN   REACTION    IN    SYPHILIS  429 

explanation  of  its  mechanism  is  still  lacking,  as  the  large  number  of 
theories  advanced  tend  to  show. 

While  lipoidal  extracts,  as  well  as  normal  and  luetic  serums,  may  sepa- 
rately absorb  or  fix  small  amounts  of  complement,  a  mixture  of  a  suitable 
extract  and  syphilitic  serum  is  capable  of  fixing  large  amounts  of  comple- 
ment, and  this  constitutes  the  main  principle  and  all  that  is  definitely 
known  of  the  syphilitic  reaction. 

The  serum  of  a  syphilitic  is  characterized,  therefore,  by  the  presence 
of  this  lipodotropic,  antibody-like  substance,  which  has  a  great  affinity 
for  lipoids  and  in  mixture  with  them  will  cause  the  absorption  or  fixation 
of  complement  to  a  well-marked  degree.  Instead  of  being  an  example 
of  complement  fixation  in  a  mixture  of  specific  antigen  with  specific 
antibody,  as  originally  believed,  it  is  technically  a  non-specific  reaction, 
but  practically  it  is  highly  specific,  since  this  peculiar  antibody  is  found 
in  largest  amount  and  most  constantly  in  syphilis,  and  to  a  lesser  extent 
in  practically  only  two  other  diseases,  namely,  leprosy  and  frambesia. 
In  countries  and  districts  where  these  diseases  are  infrequent  or  unknown 
with  proper  technic  the  reaction  for  syphilis  is  highly  specific. 

As  will  be  shown  further  on,  the  presence  of  this  lipodotropic  sub- 
stance is  dependent  upon  the  activities  of  the  Treponema  pallidum, 
and  when  repeated  tests  continue  to  show  its  presence,  there  is  every 
reason  to  believe  that  a  cure  has  not  been  effected,  but  that  the  patient 
still  harbors  the  living  parasite. 

Citron  has  advanced  the  hypothesis  that  the  antibody-producing 
antigen  is  a  toxolipoid,  which  would  explain  the  fact  that  while  pure 
lipoids,  such  as  lecithin,  cannot  stimulate  antibodies  (Bruck),  as  the 
toxolipoid  does,  they  can,  nevertheless,  react  with  the  lipodotropic 
antibodies  in  vitro,  with  fixation  or  absorption  of  complement.  As 
Sachs  and  Altman  point  out,  an  equally  tenable  theory  would  be  that  in 
syphilis  the  tissues  undergo  such  alterations  that  they  can  produce  anti- 
bodies to  the  lipoid  substances  as  may  be  contained  in  the  spirochetes 
themselves. 

While  the  production  of  this  lipodotropic  antibody  is  still  unex- 
plained, the  fact  remains,  nevertheless,  that  it  forms  the  basis  of  the 
biologic  syphilitic  reaction,  and  in  a  mixture  with  a  suitable  lipoid  is 
capable  of  absorbing  or  inactivating  complement  to  a  marked  degree. 
Whether  or  not  it  is  a  true  antibody  in  the  sense  that  it  is  inimical  to  the 
spirochete  is  doubtful;  by  many  it  is  regarded  as  a  secondary  product  of 
cellular  activity,  and  has  been  called  syphilis  "reagin." 

Since  similar  "reagins"  are  to  be  found  in  other  infections,  notably 
in  frambesia  and  leprosy,  investigators  in  this  field  anxiously  awaited 


430  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

the  isolation  of  the  Treponema  pallidum  in  pure  culture,  believing  that 
if  this  result  were  secured  it  would  be  possible  to  work  with  a  specific  anti- 
gen, determine  the  nature  of  the  true  syphilis  antibody,  and  possibly 
establish  a  complement-fixation  test  specific  for  syphilis. 

In  1909  Schereschewsky,1  using  an  antigen  of  an  impure  and  non- 
pathogenic  culture  of  a  spirochete  regarded  as  the  Treponema  pallidum, 
reported  positive  complement-fixation  reactions  with  the  majority  of 
serums  tested. 

In  1912  Noguchi,2  having  undoubtedly  isolated  the  spirochete  in 
pure  culture,  prepared  antigens  and  found  that,  whereas  certain  long- 
standing or  treated  cases  of  syphilis  yielded  positive  reactions  with  the 
pallidum  antigens,  the  reactions  were  uniformly  negative  when  the 
lipoidal  extracts  were  used.  In  primary  and  secondary  syphilis  the 
reactions  with  pallidum  antigens  were  uniformly  negative,  whereas  with 
the  lipoidal  extracts  they  were  uniformly  positive.  As  a  result  of  his 
experiments  Noguchi  concluded  that  in  syphilis  there  is  produced  a 
true  antibody  that  reacts  specifically  with  pallidum  antigen,  in  addition 
to  the  lipodotropic  "reagin,"  which  reacts  with  lipoidal  extracts,  and 
whereas  the  latter  indicates  activity  of  the  infecting  agent,  the  former 
is  a  gage  of  the  defensive  activity  of  the  infected  host. 

Craig  and  Nichols,3  using  alcoholic  extracts  of  pure  cultures  in  ascites 
kidney  agar  of  Treponema  pallidum,  Spirochete  pertenuis,  and  Spiro- 
chete microdentium,  found  similar  positive  reactions  in  all  stages  of 
syphilis  with  the  three  antigens,  but  the  reactions  were  weaker  and  less 
constant  as  compared  with  those  obtained  with  a  stock  of  lipoidal  extract. 

Similar  studies  conducted  by  Kolmer,  Williams,  and  Laubaugh4  with 
aqueous  and  alcoholic  extracts  of  pallidum  cultures  showed  positive 
reactions  in  secondary,  tertiary,  and  congenital  syphilis.  The  aqueous 
extracts  yielded  better  reactions  than  the  alcoholic  extracts;  in  practi- 
cally all  instances,  however,  the  reactions  were  weaker  than  those  ob- 
tained with  the  ordinary  lipoidal  extracts.  Control  antigens  of  typhoid 
and  cholera  bacilli  and  sterile  culture  mediums  demonstrated  that  all 
contained  lipoidal  substances  that  may  give  weak  reactions  with  the 
lipodophilic  " reagin."  This  may  explain  Schereschewsky's  positive 
reactions  with  an  antigen  of  a  spirochete  that  in  all  probability  was 
Spirochete  microdentium  (Noguchi). 

The  true  nature  of  the  Wassermann-Detre  reaction,  therefore,  cannot 

1  Deutsch.  med.  Wchnschr.,  1909,  xxxv,  1652. 

2  Jour.  Amer.  Med.  Assoc.,  1912,  Iviii,  1163. 

3  Jour.  Exper.  Med.,  1912,  xvi,  336. 

4  Jour.  med.  Research,  1913,  xxviii,  345. 


GENERAL   TECHNIC  431 

be  said  to  have  been  determined.  It  is  probable  that  the  ordinary 
syphilis  reaction  is  in  itself  not  dependent  upon  a  true  antibody,  and  that 
the  reaction  is  not  an  immunity  reaction,  but  due  rather  to  the  presence 
of  peculiar  tissue  products  (reagins)  altered  by  the  presence  and  activ- 
ities of  the  spirochetes  themselves,  and  that  the  Wassermann  reaction 
is  an  expression  of  this  active  injury  to  tissue-cells.  In  addition  to  this 
secondary  product  there  is  probably  a  true  syphilis  antibody  that  may 
yield  specific  complement  fixation  with  pallidum  antigens. 

In  so  far  as  the  Wassermann  reaction  is  concerned,  the  true  antibody 
is  entirely  secondary  in  importance,  and  the  whole  question  is  intimately 
concerned  with  the  chemistry  of  lipoids.  While  future  researches  in 
immunochemistry  may  reveal  the  mechanism  of  the  reaction,  the  prin- 
ciples are  at  least  well  understood  at  present,  so  that  the  syphilis  reac- 
tion is  proving  of  great  diagnostic  and  practical  value. 

TECHNIC  OF  THE  WASSERMANN  REACTION 
Glassware  for  Complement-fixation  Reactions. — Test-tubes  should  be 
of  convenient  sizes, — 12  by  1.5  cm., — perfectly  clean,  free  from  acids 
and  alkalis,  and  preferably  sterile.  They  need  not  be  plugged  with 
cotton  as  it  suffices  to  sterilize  them  in  a  wire  basket  with  their  mouth- 
ends  downward.  Smaller  test-tubes,  as  those  used  in  the  Noguchi 
modification  of  the  Wassermann  reaction  (8  by  1  cm.),  are  wrapped  in 
newspaper  in  bundles  of  25  and  sterilized. 

Pipets  should  be  perfectly  clean  and  preferably  sterile.  Three  kinds 
are  required:  The  ordinary  1  c.c.  pipet,  graduated  to  0.01  c.c.  and 
calibrated  to  the  tip;  a  number  of  special  pipets  for  the  fourth  method 
and  the  gonococcus  fixation  test,  of  about  the  same  length  and  external 
diameter  as  an  ordinary  1  c.c.  pipet,  but  of  much  smaller  caliber,  so 
that  the  pipet  will  hold  0.2  c.c. ;  it  should  be  graduated  to  0.01  c.c.  and 
calibrated  to  the  tip;  5  c.c.  pipets  divided  into  0.1  c.c.  Care  should  be 
exercised  in  handling  pipets  to  avoid  breaking  the  tips.  After  use  they 
should  be  washed  free  from  blood,  serum,  etc. 

GENERAL  TECHNIC 

For  testing  for  the  Wassermann  syphilis  reaction  five  reagents  are 
used: 

1.  The  fluid  to  be  tested. 

2.  Complement. 

3.  Hemolytic  amboceptor. 

4.  Blood-corpuscles. 

5.  Antigen  (organic  extract). 


432         THE   TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

I.  The  Fluid  to  be  Tested. — (a)  Serum. — As  a  general  rule,  all 
specimens  of  blood  submitted  for  complement-fixation  tests  should  be 
collected  aseptically  in  sterile  containers.  This  is  especially  necessary 
when  there  has  been  delay  in  transmitting  the  fluid  to  the  laboratory,  as 
when  sent  through  the  mails  from  distant  points.  When  the  reactions  are 
to  be  conducted  on  the  same  or  on  the  following  day,  the  specimen  of 
blood  may  be  collected  in  chemically  clean  but  not  necessarily  sterile 
containers.  Bacterial  contamination  renders  a  fluid  anticomplementary 
and  unfit  for  complement-fixation  tests.  Specimens  should  be  kept  on 
ice  until  used,  and  the  serum  promptly  separated  from  the  clot. 

Collecting  Blood  for  the  Wassermann  Reaction. — In  collecting  blood 
for  the  Wassermann  reaction  the  following  points  should  be  remembered : 

1.  That  during  active  antisyphilitic  treatment  the  blood  may  react 
negatively,  whereas  at  a  later  period  a  true  positive  reaction  is  observed. 
It  is  well,  therefore,  not  to  collect  blood  until  all  specific  treatment  has  been 
suspended  for  at  least  two  weeks. 

2.  That  blood  collected  during  or  immediately  after  an  alcoholic 
debauch  may  yield  a  false  negative  reaction  (Craig  and  Nichols). 

3.  That  blood  should  not  be  collected  just  after  anesthesia  or  while 
the  patient  has  a  high  temperature. 

As  a  general  rule,  at  least  1  c.c.  of  serum  and  2  c.c.  of  cerebrospinal 
fluid  are  required  for  making  the  syphilitic  reaction.  From  2  to  3  c.c. 
of  blood  are  needed,  these  amounts  being  easily  collected  from  adult 
.persons  by  pricking  the  finger  deeply  and  filling  a  small  test-tube  or 
vial,  as  shown  on  p.  32.  This  method  is  very  convenient,  especially 
for  physicians,  hospitals,  and  dispensaries  where  direct  access  to  a 
laboratory  can  be  had.  When  the  treatment  is  to  be  guided  by  the 
Wassermann  reaction,  a  number  of  tests  are  required,  and  patients  may 
object  to  repeated  venipuncture,  whereas  no  objections  will  be  raised 
to  simple  puncture  of  the  finger. 

Larger  amounts  of  blood  are  collected  from  a  vein  at  the  elbow  under 
aseptic  precautions,  as  described  on  p.  33.  As  a  rule,  it  is  well  to  collect 
at  least  5  c.c.  of  blood,  especially  if  the  specimen  is  shipped  from  a  distant 
point  (Fig.  108).  An  excess  of  serum  permits  the  technician  to  repeat  a 
test  when  necessary,  or  to  apply  more  than  one  method,  and  thus  at 
times  both  the  physician  and  the  patient  are  saved  the  time  and  annoy- 
ance incident  to  collecting  another  specimen  (Fig.  109). 

The  Keidel  tube,  which  is  sterilized  and  ready  for  use,  is  quite  a 
convenience  (p.  36).  However,  a  test-tube  or  a  centrifuge  tube  may  be 
used,  or,  when  a  specimen  is  to  be  mailed,  a  5  or  10  c.  c.  vial  of  thick  glass, 


GENERAL   TECHNIC 


433 


stoppered  with  a  cork  or  a  rubber  stopper  is  quite  satisfactory  (Fig.  108). 
Vial,  stopper,  and  needle  are  readily  sterilized  in  boiling  water,  drained, 
and  cooled,  the  specimen  collected,  the  vial  tightly  stoppered,  and  the 
whole  sent  at  once  to  the  laboratory.  Cotton  stoppers  are  unsatisfac- 
tory, as  unless  the  tube  or  vial  is  maintained  in  an  upright  position,  the 
fluid  may  be  absorbed.  When  specimens  of  blood  are  to  be  mailed,  it 
is  better  to  fill  a  small  vial  than  to  place  the  same  amount  in  a  large 
container,  for  in  the  latter  case  agitation  through  handling  may  result 
in  so  much  mechanical  hemolysis  taking  place 
as  to  render  the  serum  unsatisfactory  for  use. 
Specimens  so  collected  may  be  sent  for  long 
distances,  even  in  warm  weather,  and  undergo 
no  change. 

In  collecting  blood  from  children,  or  where 
the  veins  are  small,  a  proportionately  smaller 
needle  may  be  used.  In  infants  the  cupping 
apparatus  of  Blackfan  is  quite  satisfactory  (p. 
36);  frequently  sufficient  blood  may  be  ob- 
tained from  a  great  toe. 

The  specimen  of  blood  should  be  kept  in  a 
cold  place,  and  the  serum  removed  at  the  end 
of  twenty-four  hours.  Serum  that  is  allowed 
to  remain  with  the  clot  for  longer  periods  is 
more  likely  to  become  anticomplementary, 
especially  if  it  becomes  deeply  tinged  with 
hemoglobin.  In  cases  where  the  serum  does 
not  separate  the  clot  may  be  broken  up  gently 
with  a  sterile  glass  rod  and  centrifugalized. 
The  serum  should  be  clear  and  free  from  cor- 
puscles. Opalescent  and  milky  serums,  ob- 
tained  during  the  period  of  digestion  and  from 
nursing  women,  usually  do  not  interfere  with 

the  reaction;  bile-stained  serum  may  at  times  give  marked  non-specific 
fixation  of  complement. 

It  is  essential  that  all  serums  be  heated  at  55°  C.  for  half  an  hour  imme- 
diately before  the  test  is  made.  This  exposure  to  heat  somewhat  diminishes 
the  reacting  power  of  a  syphilitic  serum,  but,  as  shown  by  Seligman  and 
Pinkus,  it  is  a  necessary  procedure,  for  a  considerable  proportion  of  nor- 
mal serums  or  those  from  diseases  other  than  syphilis  will  react  positively 
when  unheated,  whereas  when  heated,  they  will  give  a  negative  reaction. 
28 


FIG.  108 — A  VIAL  TO 
CONTAIN  BLOOD  FOR 
THE  WASSERMANN  RE- 
ACTION. 

This  is  an  ordinary 
glass  vial  fitted  with  a 
rubber  stopper.  It  holds 
5  c.c.  to  the  mark,  and 
is  readily  packed  for  mail- 
ing. Never  stopper  with 
cotton.  A  good  cork  stop- 
per may  be  used. 


434        THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

Furthermore,  serums  that  are  sterile  and  are  kept  for  some  time  at 
room  temperature  or  even  in  an  ice-chest  acquire  an  anticomplementary 
power  that  is  destroyed  by  heating  at  55°  C.  for  half  an  hour.  Therefore 
when  serums  are  preserved  for  a  number  of  days  they  are  heated  not  so 
much  for  the  purpose  of  removing  native  hemolytic  complement,  which 
has  probably  already  deteriorated,  but  to  remove  thermolabile  anti- 
complement.  Serums  that  are  old  or  contaminated  with  bacteria  are  likely 
to  be  highly  anticomplementary,  and  this  property  cannot  be  destroyed 


X^\ 


FIG.  109. — OUTFIT  FOR  COLLECTING  BLOOD  FOR  THE  WASSERMANN  REACTION  (NEW 

YORK  BOARD  OF  HEALTH). 

The  container  is  a  centrifuge  tube  which  holds  about  15  to  18  c.c.  of  blood  to  the 
mark.  The  needle  is  furnished  in  a  separate  tube,  sterilized  and  ready  for  use.  This 
outfit  is  not  adapted  for  mailing. 

by  heating  at  55°  C.  or  nitration  through  a  Berkefeld  filter.  In  order  to 
.conduct  a  reliable  test  it  is  usually  necessary  to  secure  fresh  serum. 
This  anticomplementary  action  of  serums  is  so  important  that  in  every 
complement-fixation  test  there  is  a  serum  control  tube  containing  all  the 
ingredients  except  antigen,  the  object  being  to  discover  any  inhibitory  action 
of  the  serum  itself  upon  the  complement. 

Weohselmanri's  method  of  converting  syphilitic  serums  showing  a 
negative  or  weakly  positive  reaction  to  those  exhibiting  a  marked  positive 


GENERAL   TECHNIC  435 

reaction  depends  upon  removing  the  excess  of  indifferent  and  inhibiting 
serum  components  and  upon  the  destruction  or  diminution  of  the  natural 
antisheep  amboceptor  present  in  so  large  a  percentage  of  human 
serums.  As  Noguchi  and  Bronfenbrenner  have  pointed  out,  this  method 
may  likewise  remove  the  antibodies  concerned  in  the  reaction.  To  1  c.c. 
of  heated  serum  add  3.5  c.c.  of  saline  solution  and  0.5  c.c.  of  a  7  per  cent, 
suspension  of  freshly  precipitated  barium  sulphate;  shake,  and  let  it 
stand  for  one  hour  at  37°  C.;  centrifugalize,  and  pipet  off  the  diluted 
serum,  which  is  now  ready  to  be  tested  (1  c.c.  =  0.2  c.c.  of  undiluted 
serum). 

Cadaver  serums  are  likely  to  be  highly  discolored  with  hemoglobin 
and  quite  anticomplementary.  Such  serums  may  be  tested  in  half  the 
usual  dose,  and  while  the  results  are  quite  specific,  they  are  not  so  reliable 
or  constant  as  those  obtained  from  the  living. 

The  doses  of  serum  used  in  testing  for  the  syphilis  reaction  are  given 
with  each  method.  In  the  original  Wassermann  test  0.2  c.c.  was  used. 
As  a  rule,  from  0.05  c.c.  to  0.2  c.c.  of  serum  are  satisfactory;  higher  doses 
may  occasionally  show  a  stronger  positive  reaction,  but  the  serum  must 
be  perfectly  fresh  to  avoid  non-specific  complement  fixation,  and  the 
natural  antisheep  amboceptor  should  first  be  removed. 

(b)  Cerebrospinal  Fluid. — In  certain  nervous  diseases  the  cerebro- 
spinal  fluid  is  examined  for  the  syphilis  reaction.    Fluid  is  secured  by 
lumbar  puncture,  according  to  the  method  described  on  p.  37.     If  the 
specimen  contains  blood,  it  should  be  centrifuged  until  it  is  clear.    It 
should  not  be  heated  before  use,  as  it  does  not  contain  hemolytic  comple- 
ment, and  fresh  fluids  from  cases  other  than  syphilitics  do  not  react  pos- 
itively.   Cerebrospinal  fluids,  as  a  rule,  possess  weaker  deviating  powers 
than  the  corresponding  blood-serum,  and  hence  it  is  necessary  to  use 
larger  doses — at  least  0.5  to  1  c.c.,  instead  of  0.05  to  0.2  c.c.,  as  in  the 
case  of  blood-serum. 

(c)  Other  Fluids. — Positive  syphilitic  reactions  have  been  described 
as  occurring  with  milk,  pleural  and  peritoneal  exudates,  and  albuminous 
urine  (Bauer  and  Hirsh)  from  luetic  cases.     The  reactions  with  these 
substances  are  conducted  in  the  same  manner  as  with  Cerebrospinal 
fluid.     The  material  should  be  perfectly  fresh,  as  anticomplementary 
action  is  likely  to  occur. 

II.  Complement. — While  complement  is  to  be  found  in  the  fresh 
normal  serum  of  practically  all  warm-blooded  animals,  not  all  are 
suitable  for  complement-fixation  tests.  A  suitable  complement  must 
possess  two  important  properties:  (1)  Complementary  activities,  or 


436        THE    TECHNIC    OF    COMPLEMENT-FIXATION   REACTIONS 

the  power  of  activating  a  hemolytic  amboceptor,  and  (2)  fixability,  or 
the  power  of  being  " fixed"  by  antigen  and  antibody.  Noguchi  and 
Bronfenbrenner  have  studied  the  complements  of  the  dog,  sheep,  hog, 
ox,  rabbit,  and  other  animals,  and  found  that  the  complement  of  the 
guinea-pig  was  best  adapted,  from  all  standpoints,  for  the  complement- 
fixation  test.  The  complements  of  pigs  and  sheep  are  quite  fixable,  but 
their  weak  hemolytic  action  and  rapid  deterioration  render  them  unsuit- 
able for  fixation  purposes.  Rabbit  complement  is  quite  active,  but  is 
not  easily  fixable.  Kolmer,  Yui,  and  Tyau1  found  rat  complement 
fairly  well  suited  for  making  the  syphilitic  reaction  with  an 
antihuman  hemolytic  system. 

The  hemolytic  power  of  guinea-pig  complement  is  not  constant. 
In  unhealthy  animals  it  is  likely  to  be  low,  and  even  among  normal 
animals  it  may  show  some  variation.  For  this  reason  the  hemolytic 
power  of  each  serum  is  determined  by  a  method  of  titration  before 
complement-fixation  reactions  are  conducted.  Fixed  doses  of  hemolysin 
and  corpuscles  may  be  used,  and  the  amount  of  complement  necessary 
for  effecting  complete  hemolysis  may  be  determined,  or  a  fixed  dose  of 
complement  and  corpuscles  may  be  used  with  different  amounts  of 
hemolysin,  the  chief  object  being  to  adjust  all  three  factors  of  the  hemo- 
lytic system,  namely,  complement,  corpuscles,  and  hemolysin,  to  exact 
and  known  proportions. 

The  complement  in  the  serums  of  different  guinea-pigs  may  show 
considerable  variation  in  fixability.  The  amount  of  complement  in- 
hibited by  serum  alone  and  organic  extract  alone,  or  by  given  constant 
quantities  .of  serum  and  extract,  may  vary  more  markedly  than  their 
complementary  activity.  To  reduce  this  error  to  a  minimum  it  is  advis- 
able, whenever  possible,  to  use  the  pooled  serums  of  two  or  more  pigs 
for  making  complement-fixation  tests. 

Guinea-pig  complement  serum  is  collected  by  bleeding  the  animal, 
under  ether  anesthesia,  into  a  Petri  dish  or  centrifuge  tube.  The  large 
vessels  on  both  sides  of  the  neck  are  quickly  severed  with  a  pair  of 
sharp-pointed  scissors  or  a  scalpel,  care  being  exercised  not  to  sever  the 
trachea  and  esophagus.  A  funnel  is  used  for  collecting  blood  in  centrifuge 
tubes.  It  is  well  finally  to  sever  the  spinal  cord,  in  order  that  the  animal 
may  not  recover  from  the  anesthetic  and  thus  insure  a  painless  operation 
throughout.  (See  p.  46.) 

It  is  best  to  keep  the  blood  at  room  temperature  for  a  few  hours 

1  Jour.  Med.  Research,  1913,  xxviii,  483. 


FlG.    110. TlTRATION    OF    HEMOLYTIC    COMPLEMENT. 

The  tube  containing  0.4  c.c.  of  complement  is  the  smallest  amount  producing  com- 
plete hemolysis,  and  this  amount  is  the  unit. 


GENERAL   TECHNIC  437 

until  coagulation  has  occurred  and  the  serum  has  separated;  then  place 
the  whole  in  the  ice-chest  until  needed.  Blood  may  be  collected  in 
centrifuge  tubes  and  allowed  to  coagulate;  it  is  then  broken  up  with  a 
glass  rod,  centrifuged,  and  the  serum  secured  at  once;  such  complement, 
however,  is  likely  to  be  unduly  hypersensitive  to  the  anticomplement 
action  of  organic  extract  and  of  mixtures  of  extract  with  normal  serums. 
As  a  general  rule,  therefore,  it  is  good  practice  to  bleed  the  animal  late  in 
the  afternoon  preceding  the  day  on  which  the  experiment  is  to  be  made,  or 
at  least  some  hours  before  the  regular  work  of  the  day  begins.  The  serum 
should  be  clear  and  contain  no  corpuscles. 

Preservation  of  Complement.— Various  methods  have  been  advo- 
cated from  time  to  time  for  the  preservation  of  complement  serum.  Se- 
rum kept  in  a  frozen  state  will  preserve  its  complement  properties  over 
a  period  of  several  weeks.  In  my  own  experience,  complement  serum  is 
best  preserved  with  chemically  pure  sodium  chlorid.  The  sera  of 
several  guinea-pigs  are  secured  and  mixed,  and  0.425  gram  sodium 
chlorid  dissolved  in  each  10  c.c.  This  serum  is  best  preserved  in  the 
refrigerator,  sealed  in  amounts  of  1  c.c.  in  ampules;  before  use  each 
cubic  centimeter  is  diluted  with  19  c.c.  of  distilled  water,  making  a 
1  : 20  or  5  per  cent,  dilution  ( =  0.05  c.c.  undiluted  serum)  in  0.85  per 
cent,  salt  solution.  If  a  1  : 10  dilution  (=  0.1  c.c.  undiluted  serum)  is 
desired,  0.85  gram  sodium  chlorid  should  be  added  to  each  10  c.c. 
of  serum  and  each  cubic  centimeter  diluted  with  9  c.c.  of  distilled 
water. 

Complement  serum  preserved  in  this  manner  will  maintain  its 
hemolytic  and  fixing  properties  for  several  weeks;  loss  in  fixability  or 
delicacy  in  the  complement-fixation  test  is  usually  apparent  before  an 
appreciable  deterioration  in  hemolytic  activity. 

And  now  we  come  to  a  very  important  question,  namely,  the  amount 
of  complement  that  is  to  be  used  in  conducting  complement-fixation  tests. 
Many  present-day  observers  use  exactly  one  unit  of  complement  and 
one  unit  of  amboceptor.  This  is  permissible,  providing  the  complement 
is  titrated  in  the  presence  of  a  constant  dose  of  antigen  and  a  constant 
dose  of  serum,  in  order  that  due  allowance  for  the  anticomplementary 
action  of  these  may  be  made  in  the  titration.  Under  these  circum- 
stances, however,  it  is  necessary  to  titrate  each  patient's  serum  with  the 
complement,  because  one  serum  or  even  the  pooled  serums  of  different 
persons  should  not  be  taken  as  a  standard  in  the  titration,  for  two  impor- 
tant reasons:  (1)  The  patient's  serum  which  we  are  about  to  test  may 


438  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

. 

t}e  more  anticomplementary  than  the  serum  used  in  the  complement 
titration,  and  hence  when  used  in  the  main  test,  with  exactly  one  unit 
of  complement,  mild  degrees  of  inhibition  of  hemolysis  will  be  secured 
that  may  be  interpreted  as  slightly  positive  reactions;  or  (2)  the  serum 
used  in  the  complement  titration  may  contain  more  or  less  natural 
hemolytic  amboceptor  than  the  patient's  serum,  and  this  factor  exerts 
an  influence  on  the  titration,  so  that  the  unit  varies  with  different  serums. 
For  these  reasons  I  have  included  here  a  fourth  method  for  using  a 
single  unit  of  complement  under  conditions  where  the  anticomple- 
mentary action  of  each  serum  and  the  antigen  are  determined,  in  pref- 
erence to  titrating  the  complement  with  a  serum  and  using  this  unit  for 
a  number  of  other  serums  that  are  sure  to  differ  from  each  other. 

In  this  titration,  therefore,  we  determine  the  amount  or  unit  of 
complement  necessary  to  produce  hemolysis  with  fixed  amounts  of 
amboceptor  and  corpuscles.  It  will  be  observed  that  I  have  used  two 
units  of  amboceptor,  as  determined  by  a  previous  titration.  These  two 
units  are  equivalent  to  one  dose,  and  the  same  would  be  true  whether 
three,  four,  five,  or  more  units  were  used,  because  in  this  titration  the 
corpuscles  and  amboceptor  are  arbitrary  and  fixed  constants,  and  are 
used  for  determining  the  amount  of  complement  necessary  to  bring 
about  complete  hemolysis. 

In  conducting  the  main  tests  the  dose  of  corpuscles  and  that  of 
amboceptor  are  the  same  as  those  used  in  the  complement  titration,  but 
instead  of  using  exactly  one  unit  of  complement,  it  is  necessary  to  use 
one  and  one-half  or  two  units,  to  allow  for  the  anticomplementary 
action  of  antigen  and  patient's  serum. 

As  the  result  of  a  large  number  of  comparative  titrations  and  tests 
I  have  found  that  0.05  c.c.  of  complement  serum  (  =  1  c.c.  of  a  1:20 
dilution)  is  a  safe  amount  to  use  with  2.5  per  cent,  corpuscle  suspension, 
and  equally  good  results  are  secured  by  adopting  this  amount  as  a  fixed 
dose  in  titrating  the  amboceptor  before  each  day's  work.  If  the  pig 
serum  happens  to  be  weaker  or  stronger  in  complement  than  it  is  ex- 
pected to  be,  the  differences  are  adjusted  in  the  amboceptor  titration. 
Following  the  same  rule,  one  and  one-half  or  two  units  of  amboceptor 
are  used  in  conducting  the  main  test,  the  extra  half,  or  one  unit,  over- 
coming the  anticomplementary  action  of  antigen  and  patient's  serum. 

It  is  important  to  remember  that,  in  conducting  this  titration, 
amboceptor,  complement,  and  corpuscles  are  to  be  mixed  at  once;  if 
amboceptor  and  corpuscles  are  mixed  and  allowed  to  stand  for  ten 


GENERAL   TECHNIC  .     439 

minutes  or  more  before  receiving  the  complement  the  corpuscles 
become  "sensitized,"  and  the  amount  of  complement  required  for 
effecting  hemolysis  will  be  less  than  if  all  three  are  mixed  one  after 
another.  If  this  rule  is  not  adhered  to,  an  error  in  technic  may 
result. 

HI.  Hemolytic  Amboceptor. — Since  the  original  work  of  Wasser- 
mann  appeared,  the  antisheep  hemolytic  system  has  been  most  widely 
used  in  experimental  investigations. 

Antisheep  amboceptor  is  readily  prepared  by  immunizing  rabbits 
with  washed  sheep's  corpuscles.  A  simple  and  efficient  method  is  to 
give  intravenous  injections  of  four  doses  of  5  c.c.  each  of  a  10  per  cent, 
suspension  every  three  or  four  days.  Other  methods  and  the  details 
of  the  preparation  and  preservation  of  amboceptor  are  given  in  Chap- 
ters IV  and  V. 

The  one  objection  to  the  use  of  the  antisheep  hemolytic  system  is 
the  presence,  in  a  large  proportion  of  human  serums,  of  variable  amounts 
of  natural  amboceptor  for  sheep's  cells.  In  about  70  per  cent,  of  fresh 
inactivated  human  serums  sufficient  amboceptor  is  present  partially 
or  completely  to  hemolyze  the  usual  dose  of  sheep-cell  emulsion  with 
the  customary  amount  of  guinea-pig  complement.  In  fact,  the  Bauer 
and  Hecht  modifications  of  the  Wassermann  reaction  utilize  this  natural 
amboceptor,  but,  as  will  be  pointed  out  further  on,  this  factor  is  too 
variable  to  be  employed  in  conducting  the  reaction,  as  non-specific  or 
false  positive  results  are  quite  likely  to  occur. 

As  has  been  stated  in  the  preceding  chapter,  the  delicacy  and  accuracy 
of  any  complement-fixation  test  depend  to  a  large  extent  upon  proper 
adjustment  of  the  hemolytic  system.  It  will  readily  be  understood  that 
the  presence  of  an  unknown  quantity  of  natural  amboceptor  in  a  serum 
is  a  drawback  to  accurate  quantitative  estimations.  The  importance 
of  this  lies  in  the  fact  that  for  some  unknown  reason  an  excess  of  ambo- 
ceptor may  completely  hemolyze  the  corpuscles,  even  though  a  small 
amount  of  the  necessary  complement  has  been  specifically  fixed  by 
antigen  and  syphilis  antibody.  In  this  manner  negative  reactions  may 
result  with  serums  that  would  otherwise  show  a  slight  positive  reaction. 
To  remove  this  source  of  error  Noguchi  has  advocated  the  use  of  an 
antihuman  hemolytic  system,  which  renders  the  reaction  more  delicate. 
However,  comparative  studies  between  antisheep  and  other  hemolytic 
systems  demonstrate  that,  with  proper  technic,  the  influence  of  natural 
amboceptors  may  be  reduced  to  a  minimum  and  rendered  almost  neg- 


440        THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

ligible.  At  any  rate  it  is  very  simple  and  but  little  trouble  to  remove  the 
antisheep  amboceptor  routinely  from  human  serums  previous  to  mak- 
ing the  tests  (for  technic,  see  p.  400) . 

A  method  for  titrating  immune  amboceptor  has  been  given  on  p.  397. 
This  important  feature  will  be  dealt  with  again  in  giving  a  detailed 
description  of  the  various  methods  that  follow. 

IV.  Red  Blood  Corpuscles. — Defibrinated  sheep  blood  is  washed 
three  times  with  an  excess  of  sterile  normal  salt  solution  to  remove  all 
traces  of  serum  (p.  28). 

In  the  original  Wassermann  reaction  a  5  per  cent,  suspension  in  salt 
solution  is  used  in  doses  of  1  c.c.  This  emulsion  is  quite  heavy,  and 
sharper  and  clear  results  are  secured  by  using  just  half  this  amount  and 
at  the  same  time  sufficient  cells  are  used  to  make  the  readings  easy  and 
distinct.  Either  0.5  c.c.  of  a  5  per  cent,  or  1  c.c.  of  a  2.5  per  cent,  sus- 
pension may  be  used.  I  have  used  the  latter  with  entire  satisfaction  for 
several  years. 

The  emulsion  of  cells  is  prepared  with  sterile  0.85  per  cent,  sodium 
chlorid  solution.  To  2.5  c.c.  of  corpuscles  sufficient  salt  solution  is 
added  to  bring  the  total  volume  of  the  emulsion  up  to  100  c.c.,  or  smaller 
amounts  may  be  prepared  by  suspending  1  c.c.  of  corpuscles  in  39  c.c. 
of  salt  solution. 

Before  each  day's  work  the  amount  of  corpuscle  suspension  needed 
should  be  calculated,  and  sufficient  for  the  day  prepared  at  one  time,  for 
if  a  fresh  emulsion  is  prepared  later,  titration  with  the  complement  and 
amboceptor  would  be  required.  Attempts  to  count  the  corpuscles  in 
suspension  can  only  be  regarded  as  approximate  and  are  unreliable. 
By  titrating  each  emulsion  with  the  complement  and  amboceptor  to  be  used, 
that  particular  emulsion  is  thereby  adjusted,  so  that  it  is  immaterial  whether 
a  few  more  or  a  few  less  corpuscles  are  present. 

Sheep's  blood  is  obtained  either  from  an  abattoir  or  by  bleeding  an 
animal  from  the  external  jugular  vein  (p.  48).  The  latter  method  is 
preferable,  but  due  care  must  be  exercised  not  to  bleed  too  frequently 
or  in  excessive  amounts,  as  if  anemia  occurs  the  corpuscles  become 
unduly  fragile. 

Sheep's  cells  are  easily  preserved  in  a  satisfactory  condition  for 
forty-eight  hours  by  first  washing  them  and  then  storing  the  sedimented 
corpuscles  in  a  good  ice-chest.  Suspensions  are  less  easily  preserved. 
It  is  best  to  use  fresh  corpuscles,  and  those  that  are  several  days  old 
and  unduly  fragile  should  never  be  used.  Attempts  to  preserve  cor- 
puscles with  the  aid  of  mercury  bichlorid,  formalin,  etc.,  have  not  yielded 
satisfactory  results. 


GENERAL   TECHNIC  441 

V  Antigen. — As  was  previously  stated,  the  ordinary  alcoholic 
extracts  of  syphilitic  livers  used  as  " antigens"  in  conducting  the  Wasser- 
mann  reaction  are  not  biologically  specific.  It  is  generally  accepted  that 
even  in  watery  extracts  of  syphilitic  livers  the  main  antigenic  principles 
are  lipoidal  substances,  independent  of  the  Treponema  pallidum  itself. 
Next  to  pure  cultures  of  pallida,  these  aqueous  extracts  of  luetic  livers 
come  closest  to  being  a  specific  biologic  antigen.  Although  alcoholic 
extracts  of  luetic  liver  may  contain  special  lipoidal  substances  that 
enhance  their  efficacy  as  antigens,  yet,  as  shown  by  Noguchi,  and  as 
confirmed  later  by  us,  alcohol  does  not  serve  well  to  extract  pure  cultures 
of  pallida,  and  therefore  these  extracts  can  hardly  be  regarded  as  specific, 
in  the  sense  that  they  contain  antigenic  principles  of  the  spirochetes 
themselves.  The  only  specific  biologic  antigen  is  an  aqueous  extract 
of  a  pure  culture  of  pallida;  this  antigen  is,  however,  much  less  service- 
able than  an  ordinary  organic  extract,  because  the  Wassermann  reaction 
depends  upon  the  peculiar  lipodophilic  "reagin,"  which  absorbs  com- 
plement with  lipoids  in  a  characteristic  but  biologically  non-specific 
manner. 

The  term  "  antigen,"  as  ordinarily  used  in  the  Wassermann  reaction, 
must  therefore  be  regarded  as  a  misnomer.  It  is,  however,  so  generally 
used  that  it  may  be  retained,  with  a  distinct  understanding  as  to  its 
actual  meaning. 

With  the  discovery  that  alcoholic  extracts  of  normal  organs  may  serve 
as  antigen  and  that  the  chief  antigenic  principles  reside  in  the  lipoids, 
it  followed  that  extensive  researches  were  undertaken  in  the  hope  of 
discovering  a  lipoid,  or  a  combination  of  lipoids,  that  would  prove  suffi- 
ciently delicate  to  act  specifically  in  the  serum  diagnosis  of  syphilis,  and 
not  with  normal  serums  or  those  of  persons  suffering  from  other  dis- 
eases. As  a  result,  a  large  number  of  different  extracts  are  in  use.  Each 
has  its  own  advocates,  so  that  the  general  subject  of  antigens  is  the  most 
complicated  one  with  which  we  have  to  deal  in  performing  the  Wasser- 
mann reaction. 

While  various  organic  extracts  may  be  used,  practical  experience  has 
shown  that  some  are  better  than  others.  It  is  important  to  remember: 

1.  That  all  antigens  are  capable  in  themselves  of  absorbing  a  certain, 
amount  of  complement.  This  is  due  to  the  presence  of  undesirable 
extractives,  some  preparations  containing  more  than  others.  In  certain 
doses,  however,  all  antigens  are  capable  of  exerting  this  anticomple- 
mentary  action,  and,  other  things  being  equal,  that  antigen  is  best  which 
shows  this  non-specification  in  the  smallest  degree.  Before  an  antigen 


442        THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

may  be  used  in  conducting  any  complement-fixation  test  it  is  necessary 
to  ascertain  its  anticomplementary  dose,  for  if  this  dose  were  used,  a 
portion  of  or  all  the  complement  would  be  fixed  in  a  non-specific  manner, 
so  that  hemolysis,  being  partial  or  absent,  yields  false  positive  reactions.  j 

2.  Most  antigens,  when  in  sufficiently  large  amounts,  are  hemolytic,  \/ 
i.  e.,  they  may  hemolyze  corpuscles  in  a  non-specific  manner.     This 
hemolytic  action  is  usually  due  to  the  presence  of  undesirable  extractives, 
and  extracts  of  organs  that  have  undergone  advanced  autolysis  or  fatty 
degeneration  are  known  to  contain  more  of  these  hemolytic  substances 
than  do  extracts  of  normal  organs.     As  a  general  rule,  a  highly  anti- 
complementary  antigen  is  likely  to  be  correspondingly  highly  hemolytic. 
The  hemolysis  may  be  due  to  the  presence  of  lipoidal  substances  or  to 
the  alcohol  used  in  preparing  the  extract.    If  an  antigen  were  used  in  an 
amount  equal  to  its  hemolytic  dose,  partial  or  complete  hemolysis  would 
occur  in  all  tubes,  so  that  a  false  negative  result  would  be  secured.    As 

a  rule,  the  hemolytic  dose  of  an  antigen  as  determined  by  titration  in 
the  presence  of  serum  is  larger  than  the  anticomplementary  dose,  so  that 
if  the  latter  is  known,  it  is  not  always  necessary  to  determine  the  former.  . 

3.  Practically  every  alcoholic  organic  extract  will  serve,  in  certain   v 
amounts,  to  absorb  complement  in  the  presence  of  the  serum  of  a  syphilitic 
person.    Some  extracts,  however,  will  do  this  better  than  others.    The 
Wassermann  reaction  depends  upon  the  fact  that  a  larger  amount  of 
complement  is  fixed  by  the  syphilis  antibody  and  extract  than  is  fixed 
by  normal  serum  or  the  serum  of  a  person  with  some  disease  other  than 
syphilis  and  this  same  extract.    The  only  two  notable  exceptions  to  this 
general  rule  are  to  be  found  with  the  serum  of  tuberous  leprosy  and  that 

of  frambesia.  The  amount  of  antigen  that  is  found,  by  a  process  ofv> 
titration,  to  fix  a  large  amount  of  complement  with  a  constant  dose  of 
syphilitic  serum  is  known  as  its  antigenic  dose.  Not  every  lipoid  serves 
equally  well  as  antigen,  and  therefore  considerable  research  work  has 
been  done  in  the  hope  of  discovering  an  extract  or  a  combination  of 
lipoidal  substances  that  would  show  a  constant  reaction  and  would  react  . 
only  with  the  syphilis  antibody.  Thus  far  this  has  not  been  accom- 
plished; unfortunately,  pure  pallida  antigens  are  not  entirely  specific  or 
serviceable,  and  if  the  specific  and  ideal  antigen  is  discovered  in  the  future 
it  will  probably  be  of  the  nature  of  a  lipoidal  substance,  altered  or  pro- 
duced in  a  specific  manner  by  the  Treponema  pallidum  itself.  In  the 
meantime  we  have  antigens  sufficiently  delicate  and  specific,  when 
properly  used,  to  render  the  Wassermann  reaction  of  great  value  in  the 
diagnosis  of  syphilis  and  to  serve  as  a  guide  to  its  treatment. 


GENERAL   TECHNIC  443 

From  a  practical  standpoint,  therefore,  to  be  suitable  for  use  as 
antigen  in  the  syphilitic  reaction  any  extract  or  preparation  must  fulfill 
the  following  requirements: 

1.  It  should  be  largely  free  from  anticomplementary  action. 

2.  It  should  likewise  be  free  from  hemolytic  action,  in  small  doses  at 
least. 

3.  It  should  possess  a  high  degree  of  sensitiveness  for  the  syphilitic 
antibody,  i.  e.,  be  capable  of  absorbing  relatively  large -amounts  of  com- 
plement in  the  presence  of  syphilitic  serum.    A  good  antigen  is  one  that, 
in  small  amounts,  is  perfectly  antigenic,  and  that  does  not  become 
anticomplementary  or  hemolytic   until  from  four  to  ten  times  this 
amount  is  used. 

4.  It  should  be  quite  stable  and  not  difficult  to  prepare,  and  different 
preparations  should  bear  a  certain  relationship  to  one  another  in  their 
properties — that  is  they  should  keep  well,  and  different  extracts  prepared 
in  the  same  manner  should  show  fairly  constant  antigenic,  anticomple- 
mentary, and  hemolytic  doses. 

Preparation  of  Antigens. — The  following  antigens  have  been  most 
widely  used  and  recommended: 

1.  Aqueous  extract  of  syphilitic  livers. 

2.  Alcoholic  extracts  of  syphilitic  liver. 

3.  Alcoholic  extracts  of  normal  organs. 

4.  Alcoholic  extracts  of  normal  organs  reenforced  with  cholesterin. 

5.  Acetone-insoluble  lipoids. 

6.  Lecithin  and  cholesterin. 

7.  Aqueous  extracts  of  pallidum  culture. 

1.  Aqueous  Extracts  of  Syphilitic  Livers. — This  is  the  original  anti- 
gen, as  employed  by  Wassermann,  Neisser,  and  Bruck;  Wassermann  still 
uses  these  extracts  in  preference  to  others.  They  may  contain  spiro- 
chetes  or  their  direct  derivatives,  and,  as  shown  originally  by  Wasser- 
mann and  Neisser,  may  be  true  biologic  antigens,  for  when  injected  into 
monkeys,  antibodies  are  formed. 

No  satisfactory  analyses  of  these  extracts  have  been  made.  Chem- 
ically they  differ  in  no  essential  respect  from  the  liver  of  acute  yellow 
atrophy  (Ehrmann  and  Stern;  Seligman  and  Pinkus).  As  antigen  they 
are  more  efficient  than  similar,  extracts  of  normal  liver.  The  nature  of 
the  specific  factor  has  not  yet  been  demonstrated  with  certainty.  They 
react  with  the  serums  of  leprosy  and  yaws,  and,  as  in  the  case  of  other 
antigens,  their  main  antigenic  principle  is  apparently  due  to  the  presence 
of  lipoids. 


444        THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

They  are  less  stable  than  alcoholic  extracts,  and  are  likely  to  become 
highly  anticomplementary  and  lose  their  power  of  reacting  with  syphil- 
itic serums.  Citron  is  convinced  that  these  changes  are  brought  about 
by  careless  handling  of  the  extract,  or  its  exposure  to  the  light.  He 
recommends  that  the  extract  be  kept  constantly  in  the  ice-chest,  and 
that  it  be  kept  out  only  long  enough  to  remove  sufficient  for  the  day's 
work. 

Preparation. — The  fresh  liver  taken  from  a  syphilitic  fetus,  and  showing  the 
presence  of  spiroehetes  by  dark-ground  illumination,  is  weighed  and  cut  into  fine 
pieces.  Four  times  its  weight  of  0.5  per  cent,  phenol  in  physiologic  salt  solution  is 
added.  The  mixture  is  placed  in  a  brown  bottle  and  shaken  mechanically  at  room 
temperature  for  twenty-four  hours.  It  is  then  filtered  through  gauze,  to  remove 
the  larger  particles,  and  stored  in  a  brown  bottle  in  an  ice-chest.  After  several  days 
of  sedimentation  the  fluid  assumes  a  yellowish-brown  opalescence  and  is  ready  for  the 
preliminary  titration  to  determine  its  anticomplementary  and  hemolytic  doses.  The 
sediment  should  not  be  disturbed,  but  the  supernatant  fluid  should  be  carefully  re- 
moved by  means  of  a  pipet.  According  to  Citron,  extracts  that  must  be  used  in 
quantities  of  less  than  0.1  c.c.  are,  as  a  general  rule,  unsatisfactory.  Only  such  extracts 
should  be  used  as  in  doses  of  0.4  c.c.  will  not  interfere  with  hemolysis.  The  method 
of  making  these  titrations  is  given  on  page  452. 

2.  Alcoholic    Extracts    of    Syphilitic   Livers. — These    antigens    are 
extensively  used.    They  are  not  true  biologic  antigens,  for  they  do  not 
give  rise  to  antibodies  (Schatilof  and  Isabolinsky;  Seligman  and  Pinkus) ; 
they  are,  however,  usually  better  antigens   than  similar  extracts  of 
normal  liver,  a  fact  that  may  be  explained,  in  part  at  least,  by  chem- 
ical changes,  namely,  fatty  changes,  autolysis,  soaps  (Beueker),  excess 
of  cholesterin  (Piglini),  etc.,  which,  while  not  specifically  syphilitic  in 
nature,  are  often  produced  to  a  striking  degree  in  congenital  syphilis. 

Preparation, — Fetal  liver  known  to  contain  numerous  spiroehetes  is  used  in 
preparing  this  extract.  Fresh  organs  may  be  examined  at  once  by  dark-field  illu- 
mination, or  if  this  is  impossible  and  the  fetus  shows  signs  of  syphilis,  the  liver  may  be 
cut  into  large  pieces  and  preserved  in  70  per  cent,  alcohol.  After  a  few  days  a  section 
is  removed  and  stained  by  the  Levaditi  method  for  spiroehetes.  If  these  microorgan- 
isms are  numerous,  the  liver  is  suitable  for  preparing  the  antigen;  otherwise  it  should 
be  discarded.  Very  fatty  livers  are  to  be  avoided,  and  those  of  still-born  fetuses  are 
to  be  preferred. 

Ten  grams  of  liver  are  minced,  ground  with  quartz  sand,  and  treated  with  100 
c.c.  of  absolute  ethyl  alcohol.  The  mixture  is  shaken  mechanically  with  glass  beads 
for  twenty-four  hours,  and  extracted  in  the  incubator  for  ten  days.  The  containing 
flask  or  bottle  should  be  well  stoppered  to  prevent  undue  evaporation,  and  should  be 
shaken  up  at  least  once  a  day.  The  extract  is  then  filtered  through  fat-free  paper  or 
paper  washed  with  ether  and  alcohol  to  remove  the  hemolytic  substances  that  may 
be  present.  The  filtrate  is  measured,  and  the  loss  by  evaporation  is  made  up  by 
the  addition  of  more  alcohol.  If  a  shaking  apparatus  is  not  at  hand,  extractions  may 
be  left  in  the  incubator  a  few  days  longer.  After  standing  a  few  days  a  sediment 
forms,  which  should  not  be  removed  or  disturbed. 

3.  Alcoholic  Extracts  of  Normal  Organs. — These  are  used  extensively, 
at  present,  and  apparently  yield  results  equal  to  those  obtained  with 
extracts  of  luetic  liver.    It  is  certainly  true  that  a  good  extract  of  a  normal 
organ  is  superior  to  a  poor  one  prepared  from  luetic  liver.    Many,  with 


GENERAL   TECHNIC  445 

the  idea  of  specificity  uppermost  in  their  mind,  adhere  to  the  use  oi  the 
latter,  whereas  the  results  of  research  and  of  practical  work  shows  that 
lipoids  from  normal  organs  serve  equally  well  as  antigen  in  making  the 
syphilitic  reaction,  and,  indeed,  may  prove  superior  if  luetic  liver  is 
used  that  has  undergone  advanced  fatty  changes  or  autolysis,  when 
undesirable  hemolytic  and  anticomplementary  derivatives  are  extracted 
in  excess. 

Human,  guinea-pig,  and  beef-heart  muscle  are  usually  employed. 
The  first  is  especially  efficient  and  is  to  be  preferred. 

Preparation. — The  organ  is  obtained  fresh  from  the  autopsy  room.  It  is  freed 
from  fat,  and  to  each  10  grams  of  minced  muscle  100  c.c.  of  absolute  ethyl  alcohol 
are  added.  Extraction  is  conducted  in  exactly  the  same  manner  as  was  described  in 
the  preparation  of  alcoholic  extract  of  luetic  liver. 

If  guinea-pig  heart  is  employed,  as  much  of  the  fat  as  possible  should  be  removed, 
otherwise  the  extract  may  be  quite  anticomplementary. 

Boas  prepared  an  extract  of  human  heart  by  treating  the  ground  muscle  with 
nine  parts  of  absolute  alcohol,  shaking  for  an  hour  at  room  temperature,  filtering,  and 
storing  away  in  a  stoppered  bottle.  He  found  that  different  extracts  so  prepared  are 
remarkably  constant  in  their  properties,  although  they  deteriorate  rapidly  and  should 
be  prepared  freshly  every  few  weeks. 

4.  Alcoholic  Extracts  of  Normal  Organs  Reenforced  with  Choles- 
terin. — Sachs  advocated  the  addition  of  pure  cholesterin  to  alcoholic 
extracts  of  normal  heart  as  a  means  of  rendering  these  antigens  more 
delicate,  without  materially  increasing  their  anticomplementary  and 
hemolytic  properties.  He  found  that  such  preparations  possess  proper- 
ties equal  to  the  best  syphilitic  extracts.  This  work  has  been  confirmed 
by  Hemlein,  Altman,  Mclntosh  and  Fieldes,  Desmonliere,  Walker, 
and  Swift.  We  have  studied  the  subject  with  much  interest,  comparing 
the  results  with  those  secured  from  other  antigens,  as  alcoholic  extracts 
of  syphilitic  liver  and  acetone-insoluble  lipoids.  It  is  true  that  these 
preparations  are  highly  sensitive — so  much  so  that  I  never  employ  them 
alone  in  making  diagnostic  reactions,  but  always  in  conjunction  with 
other  extracts  as  controls,  in  order  to  detect  and  avoid  non-s'pecific 
reactions  with  non-luetic  serums.  We  have  found  that  they  occasion- 
ally give  faint  positive  reactions  with  normal  serums;  on  the  other  hand, 
not  infrequently,  they  react  strongly  positive  in  cases  where,  with  other 
extracts,  the  reactions  are  negative;  in  the  majority  of  such  cases 
the  serum  is  from  a  long-standing  or  a  treated  case  of  lues  that  needs 
further  treatment  until  the  reaction  with  a  cholesterin  extract  becomes 
negative.  These  alcoholic  extracts  of  normal  organs  have  their  greatest 
value,  therefore,  with  known  syphilitic  serums  when  the  reaction  is 
conducted  as  a  guide  to  treatment.  In  diagnostic  reactions,  however,  it 
is  my  opinion  that  they  should  not  be  used  alone,  but  together  with  less 


446         THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

sensitive  antigens.  In  other  words,  one  should  use  every  precaution  and 
exercise  great  care  before  making  a  diagnosis;  when  lues  is  known  to  be 
present,  however,  the  treatment  should  be  thorough,  and  there  would  seem  to 
be  no  better  criterion  for  judging  the  state  of  the  infection  than  repeated 
negative  reactions  with  cholesterin  extracts. 

Preparation. — These  extracts  are  prepared  of  human,  ox,  and  guinea-pig  heart. 
Human  heart  usually  yields  the  best  extract.  Care  should  be  taken  to  use  only 
muscle  and  to  avoid  fat.  To  10  grams  of  minced  muscle  add  100  c.c.  of  absolute 
ethyl  alcohol.  Shake  in  a  mechanical  shaker  for  twenty-four  hours,  and  continue 
the  extraction  in  the  incubator  for  ten  days  or  two  weeks.  Then  filter  through  fat- 
free  filter-paper,  and  add  absolute  alcohol  to  make  up  for  the  loss  through  evapora- 
tion. Add  0.4  gram  of  Kahlbaum's  cholesterin  (0.4  per  cent.);  shake  well  and  stand 
aside  in  the  refrigerator  for  a  few  days.  The  cholesterin  goes  into  solution  slowly  in 
cold  alcohol,  and  0.4  per  cent,  of  cholesterin  usually  saturates  the  solution.  After 
a  week  the  extract  may  be  again  filtered  and  stored  in  a  tightly  stoppered  bottle.  The 
slight  sediment  that  may  form  should  not  be  disturbed. 

These  extracts  keep  fairly  well.  Different  preparations  are  quite 
similar  in  their  properties;  they  are  usually  found  to  be  highly  antigenic 
and  no  more  anticomplementary  than  crude  alcoholic  extracts.  They 
constitute,  therefore,  inexpensive  and  very  sensitive  antigens. 

5.  Acetone-insoluble  Lipoids. — As  previously  stated,  crude  alco- 
holic extracts  may  contain  an  excess  of  undesirable  constituents,  such 
as  neutral  fats,  fatty  acids,  soaps,  and  certain  protein  materials,  which 
are  responsible  for  the  untoward  anticomplementary  and  hemolytic 
effects.  To  eliminate  these  Noguchi  advised  the  exclusive  use  of  the 
acetone-insoluble  fraction  instead  of  the  entire  unfractionated  alcoholic 
extract,  especially  if  unheated  human  serums  are  used  in  conducting  the 
syphilis  reaction.  These  extracts  are  composed  essentially  of  lecithins, 
which,  when  prepared  from  any  one  source,  consist  of  a  mixture  of 
analogous  bodies;  lecithins  from  different  sources  vary  in  their  compo- 
sition. In  speaking  of  lecithins,  one  is  prone  to  regard  them  as  chemicals, 
and  to  overlook  their  biologic  properties.  Noguchi  no  longer  employs  the 
term  lecithin  to  designate  the  acetone-insoluble  fraction  of  tissue  lipoids. 

These  antigens  are  readily  prepared  of  ox-heart  or  of  human  liver, 
the  former  being  preferable  for  use.  Their  main  disadvantage  is  the 
expense  of  preparation,  for  it  may  be  necessary  to  prepare  several 
extracts  before  one  that  is  satisfactory  is  secured.  A  good  extract  will, 
however,  keep  well,  and  is  a  reliable  and  valuable  antigen  for  the  testing 
for  the  syphilitic  reaction. 

Preparation. — A  mashed  paste  of  the  muscle  of  ox-heart  is  extracted  with  10 
parts  of  absolute  alcohol  at  37°  C.  for  four  days.  It  is  then  filtered  through  filter- 
paper  and  the  filtrate  collected  and  brought  to  a  state  of  dryness  by  evaporation. 
The  use  of  the  electric  fan  is  not  necessary,  for  if  poured  into  large  flat  dishes,  the 
filtrate  will  evaporate  in  from  twelve  to  twenty-four  hours.  The  residue  is  then 
taken  up  with  a  sufficient  quantity  of  ether,  and  the  turbid  ethereal  solution  is  allowed 


GENERAL  TECHNIC  447 

to  stand  for  a  few  hours  in  a  cool  place  until  cleared.  The  clear  ethereal  portion  is 
then  carefully  decanted  off  into  another  clean  evaporating  dish,  and  then  allowed  to 
become  concentrated  by  evaporating  the  ether  off.  The  concentrated  ethereal 
solution  is  now  mixed  with  about  10  volumes  of  pure  acetone.  A  light  yellow  precipi- 
tate forms,  which  is  allowed  to  settle,  and  the  supernatant  fluid  is  decanted  off.  Dis- 
solve each  0.3  gm.  of  this  substance  in  1  c.c.  of  ether  and  add  9  c.c.  of  pure  methyl 
alcohol.  As  a  rule,  the  greater  part  of  the  substance  goes  into  solution.  This  alco- 
holic solution  remains  unaltered  for  a  long  time,  and  is  kept  as  a  stock  solution  from 
which  the  emulsion  for  immediate  use  may  be  prepared  at  any  time  by  mixing  1  c.c. 
with  19  c.c.  of  saline  solution.  This  solution  is  then  titrated  for  antigenic,  anticom- 
plementary,  and  hemolytic  action. 

According  to  Noguchi,  if  the  extract  is  anticomplementary  or  hemo- 
lytic in  doses  of  0.4  c.c.  of  a  1  :  10  dilution,  it  is  unsuitable.  If  it  produces 
complete  inhibition  of  hemolysis  with  0.1  c.c.  of  syphilitic  serum  in  doses 
of  0.02  c.c.  or  less  of  the  same  dilution  (  =  0.2  c.c.  of  a  1:100  dilution),  it 
is  suitable.  In  making  the  fixation  test,  0.1  c.c.  of  a  1 :10  emulsion  is  to 
be  used,  thus  employing  five  times  the  minimal  antigenic  dose  which 
does  not  cause  non-specific  fixation  and  is  not  unduly  sensitive. 

6.  Lecithin  and  Cholesterin. — Browning,  Cruickshank,  and  McKenzie 
advocate  the  use  of  a  mixture  of  ox-liver  lecithin  and  cholesterin  as 
antigen  in  testing  for  the  syphilitic  reaction.  They  find  that  the  amount 
of  complement  fixed  by  lecithin  alone  with  syphilitic  serums  is  very 
much  increased  by  the  addition  of  cholesterin;  that  cholesterin  does  not 
increase  the  anticomplementary  action  of  the  antigen;  that  the  binding 
power  or  antigenic  value  of  a  mixture  of  lecithin  and  cholesterin  is  equal 
in  most  cases  to  crude  alcoholic  extracts,  but  is  less  anticomplementary. 
These  observers  use  this  mixture  in  their  modified  method,  which  con- 
sists in  the  accurate  estimation  of  the  amount  of  complement  fixed  by 
extract  and  syphilitic  serum.  It  would  seem  that  this  extract  should, 
in  the  ordinary  methods,  be  controlled  with  less  sensitive  antigens,  and 
I  have  found  that  ordinary  cholesterinized  alcoholic  extracts  of  human 
heart  are  equally  efficient  and  less  difficult  to  prepare  in  performing  a 
quantitative  Wassermann  reaction  after  the  method  just  outlined. 

Preparation. — Minced  ox-liver,  obtained  within  three  or  four  hours  after  death, 
is  digested  with  four  parts  of  95  per  cent,  alcohol  for  three  or  four  days  at  room 
temperature,  during  which  time  the  mixture  is  stirred  up  at  least  once  a"  day.  The 
filtrate  is  evaporated  in  an  open  flat  porcelain  dish  on  the  water-bath  at  60°  C.  for 
four  or  five  hours  until  a  syrupy  mass  remains.  This  is  rubbed  up  with  washed  and 
dried  quartz  sand  until  a  firm  mass  results.  The  mixture  of  dried  extract  and  sand 
(about  50  grams  of  sand  to  the  residue  of  1000  c.c.  of  extract)  is  placed  in  a  spheric 
flask,  closed  with  a  perforated  rubber  stopper  through  which  runs  a  short  piece  of 
quill  tubing  drawn  out  to  a  capillary  point  at  the  end.  This  tube  serves  to  prevent 
the  vapor  in  the  flask  from  forcing  out  the  stopper.  Ethyl  acetate  is  placed  in  the 
flask,  which  is  then  stoppered  and  immersed  up  to  its  neck  in  water  at  60°  C.  The 
flask  is  shaken  repeatedly,  and  after  ten  minutes  the  ethyl  acetate  is  poured  off  into 
a  hot-water  filter,  the  funnel  jacket  being  kept  at  60°  C.,  and  the  solution  filtered 
through  fat-free  filter-paper.  Another  portion  of  ethyl-acetate  is  added  to  the  sand, 
and  the  extraction  repeated.  After  a  third  treatment  practically  all  the  soluble  matter 
will  have  been  extracted.  In  all,  about  170  c.c.  of  ethyl  acetate  should  be  used  to 


448        THE   TECHNIC    OF   COMPLEMENT-FIXATION   KEACTIONS 

extract  the  residue  of  1000  c  c.  of  crude  extract.  The  portion  that  is  precipitated  when 
the  ethyl  acetate  solution  is  placed  in  the  ice-chest  overnight  is  again  dissolved  in 
ethyl  acetate  at  60°  C.  and  the  solution  again  placed  in  the  ice-chest  overnight. 
Finally  the  portion  insoluble  in  ethyl  acetate  in  the  cold  is  dissolved  in  water-free 
ether  (sp.  gr.,  0.717)  at  room  temperature.  To  the  ethereal  solution  in  a  glass  cyl- 
inder four  volumes  of  acetone  are  added,  causing  a  precipitate  to  be  deposited. 
Precipitation  is  aided  by  shaking  the  mixture  for  several  minutes.  Separation  is 
complete  in  ten  minutes.  The  supernatant  fluid  is  poured  off,  and  the  crude  precipi- 
tate of  lecithin  is  redissolved  in  ether  and  the  precipitation  with  acetone  repeated 
twice.  The  precipitate  is  finally  rubbed  up  with  sand  and  the  soluble  portion  taken 
up  by  extraction  with  absolute  ethyl  alcohol  for  twenty-four  hours  at  room  temper- 
ature. The  last  traces  of  lecithin  may  be  removed  by  extracting  the  residue  with  an 
additional  small  quantity  of  alcohol.  The  ordinary  commercially  "pure"  reagents 
are  satisfactory  for  this  method  of  preparation  of  lecithin. 

The  lecithin  of  1000  c.c.  of  crude  extract  should  be  extracted  with  about  100  c.c. 
of  alcohol.  The  strength  of  the  solution  is  estimated  by  evaporating  10  c.c.  at  57° 
C.  and  weighing.  The  alcoholic  lecithin  solution  is  kept  in  a  stoppered  bottle  at  room 
temperature  in  the  dark.  After  allowing  it  to  stand  for  a  week  a  0.75  per  cent, 
solution  in  alcohol  is  diluted  1 : 7  with  normal  salt  solution,  to  secure  the  maximum 
turbidity,  and  titrated.  Browning  and  McKenzie  use  0.6  c.c.  of  this  emulsion  as  the 
antigenic  dose. 

7.  Aqueous  Extract  of  Pallidum  Culture.  This  antigen  is  prepared 
by  Noguchi,  who  uses  pure  cultures  of  Treponema  pallidum  in  ascites 
kidney  agar.  Preferably  several  strains  should  be  used  in  the  prepara- 
tion, which  corresponds  quite  closely  to  luetin.  Cultures  grown  seven, 
fourteen,  twenty-one,  twenty-eight,  thirty-five,  and  forty-two  days  are 
chosen  and  examined,  and  those  that  show  the  best  growths  in  the  agar 
columns  are  selected.  The  oil  is  poured  off,  the  tubes  cut  just  above  the 
kidney,  and  the  column  of  ascites  agar  between  the  piece  of  kidney  and 
the  oil  removed  with  particular  care,  so  as  not  to  include  the  kidney  or 
the  oil.  This  substance  is  ground  in  a  mill  until  the  spirochetes  show 
disintegration.  The  thick  emulsion  is  then  diluted  with  normal  salt 
solution  and  heated  to  60°  C.  for  one-half  hour;  0.4  per  cent,  phenol  is 
added  as  a  preservative,  and  the  emulsion  titrated  for  its  anticomple- 
mentary  dose.  In  conducting  complement-fixation  reactions  with 
pallidum  antigen  one-half  of  the  anticomplementary  dose  is  used,  and  the 
serum  must  be  inactivated. 


COMPARATIVE  ANTIGENIC  VALUES  OF  VARIOUS  EXTRACTS 
For  several  years  past  I  have  been  particularly  interested  in  studying, 
from  a  practical  standpoint,  antigenic  values  of  the  extracts  most  com- 
monly employed  in  the  serum  diagnosis  of  syphilis,  and  comparing  them 
with  suitable  alcoholic  extracts  of  syphilitic  liver  as  a  standard  antigen. 
For  this  purpose  antigens  were  carefully  chosen  after  titration,  and 
only  those  were  employed  that  were  safely  free  from  anticomplementary 
action  and  whose  antigenic  dose  was  known.    A  large  number  of  serums 
and  cerebrospinal  fluids  from  syphilitic  and  non-syphilitic  persons  were 


GENERAL  TECHNIC 


449 


tested  with  numerous  different  extracts  at  the  same  time,  and  under 
similar  conditions.  A  suitable  alcoholic  extract  of  syphilitic  liver  was 
always  included  among  the  antigens  in  testing  each  serum  or  fluid,  and 
the  other  extracts  compared  with  it  in  determining  their  antigenic  value. 
In  the  following  table  the  results  of  such  studies,  covering  a  period 
of  two  years,  are  given: 

TABLE  12.— COMPARATIVE  ANTIGENIC  VALUES  OF  VARIOUS  TISSUE 

EXTRACTS 


EXTRACTS 

ANTIGENIC  PROPERTIES  AS  COMPARED  TO  ALCOHOLIC  EX- 
TRACTS OP  SYPHILITIC  LIVER 

Equal 

Stronger 

Weaker 

Negative 
(Positive 
with  Alco- 
holic Extract 
of  Syphilitic 
Liver) 

Positive 
(Negative 
with  Alco- 
holic Extract 
of  Syphilitic 
Liver) 

Cholesterinized  alcoholic  ex- 
tracts of  human,  pig,  and 
beef  heart  .  .  

50.0 
73.0 

71.1 
68.7 
73.2 

30.0 
10.8 

1.9 
4.4 

9.7 

13.4 
18.9 
23.5 

3.2 
5.7 
6.6 
3.5 

20.0 
1.8 

7.6     J 
1.3 

Acetone-insoluble  lipoids  .  .  . 
Alcoholic  extract  of  pig  and 
beef  heart  

Acetone  extract  of  syphilitic 
liver 

Alcoholic  extract  of  normal 
liver 

The  results  of  these  studies  have  shown : 

1.  That  cholesterinized  alcoholic  extracts  of  human,  beef,  and  guinea-  <- 
pig  heart  are  far  more  sensitive  than  simple  alcoholic  extracts  of  syphil- 
itic liver.    Another  peculiar  feature  of  these  antigens  is  the  fact  that  in 
syphilis,  if  they  react  at  all,  they  usually  do  so  quite  strongly. 

2.  That  the  addition  of  cholesterin  to  crude  alcoholic  extracts  of 
syphilitic  liver  and  of  normal  liver  doubles  their  antigenic  sensitiveness 
without  materially  increasing  their  anticomplementary  and  hemolytic 
action. 

3.  We  have  practically  never  found  a  serum  that  reacted  negatively 
with  a  cholesterin  extract  and  positively  with  an  alcoholic  extract  of 
syphilitic  liver.    On  the  other  hand,  in  about  20  per  cent,  of  cases  the 
cholesterinized  antigens  will  react  positively,  whereas  with  the  plain 
antigen  of  syphilitic  liver  the  reactions  are  negative.    In  the  majority 
of  such  instances  the  person  was  known  to  be  luetic,  but  had  received 
treatment  and  was  regarded  clinically  as  cured,  or  the  serum  was  that 
of  a  long-standing  and  unrecognized  case  of  lues.    Unfortunately,  slight 

29 


450         THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

reactions  may  be  secured  with  about  5  per  cent,  of  normal  serums.  For 
this  reason,  when  conducting  tests  for  diagnostic  purposes,  I  control  the 
cholesterinized  extracts  with  less  sensitive  antigens,  such  as  alcoholic 
extract  of  syphilitic  liver  and  acetone-insoluble  lipoids. 

4.  It  is  highly  important  that  these  extracts  be  carefully  standardized 
and  that  any  serum,  even  if  but  slightly  anticomplementary,  be  dis- 
carded. 

5.  In  our  experience,  repeated  negative  reactions  with  satisfactory 
cholesterinized  antigens  constitute  the  best  evidences  of  the  absence  of 
lues  or  testify  to  the  recovery  from  a  luetic  infection.    The  treatment  of 
syphilis  should  be  continued  until  the  patient's  serum  reacts  negatively 
with  alcoholic  extract  of  syphilitic  liver,  and  finally  with  cholesterinized 
extracts.    The  disease  cannot  be  regarded  as  cured  until  the  reaction  i 
has  remained  negative  for  a  year  or  two  at  least,  and  treatment  must  not 
be  discontinued  until  this  result  is  secured,  or  it  is  shown  that  the  se- 
rum is  "  Wassermann  fast"  and  that  it  is  impossible  to  secure  a  nega- 
tive reaction. 

6.  For  the  less  experienced  worker,  or  when  but  one  antigen  is  being 
used  in  conducting  the  reaction,  a  properly  prepared  alcoholic  extract  of 
syphilitic  liver  is  to  be  recommended.    One  drawback  to  the  use  of  this 
extract  is  the  difficulty  of  obtaining  suitable  tissues  for  the  preparation 
of  the  antigen.    It  has  been  my  practice  for  many  years  to  preserve  the 
livers  of  as  many  still-born  fetuses  as  I  could  obtain  in  70  per  cent, 
alcohol,  and  to  discard  them  later  unless  on  section  they  showed  the 
presence  of  numerous  spirochetes.     These  antigens  are  usually  more 
sensitive  than  similar  extracts  of  normal  liver,  but  it  is  important  to 
remember  that  not  every  extract  is  satisfactory  simply  because  it  is 
prepared  of  syphilitic  tissues. 

7.  A  suitable  preparation  of  acetone-insoluble  lipoids,  prepared  after 
the  method  of  Noguchi,  constitutes  a  sensitive,  reliable,  and  satisfactory 
antigen.    When  properly  titrated  and  standardized  and  used  with  inac- 
tivated serums,  this  antigen  may  prove  quite  sensitive  and  safe.    Nogu- 
chi, in  his  efforts  to  simplify  the  technic  of  the  syphilis  reaction,  impreg- 
nated filter-paper  with  this  antigen  and  allowed  it  to  dry.    This  prepara- 
tion is  unstable  and  generally  unsatisfactory.    The  antigen  is  best  pre- 
served in  a  stock  bottle  or  in  ampules,  and  is  diluted  with  salt  solution 
just  before  being  used  in  the  test.    Under  these  conditions  we  have  found 
these  extracts  to  be  quite  stable. 

4.  Plain  alcoholic  extract  of  human,  guinea-pig,  and  beef  heart  are 
easily  prepared,  are  quite  inexpensive,  and  when  properly  standardized 


GENERAL   TECHNIC  451 

serve  as  satisfactory  antigens.  Boas  uses  alcoholic  extracts  of  human 
heart  (Michaelis)  exclusively,  and  has  found  that  they  yield  better 
results  than  alcoholic  extracts  of  syphilitic  liver.  Garbat  and  others 
use  and  recommend  similar  extracts  of  guinea-pig  heart. 

8.  Aqueous  extracts  of  pure  cultures  of  pallida  have  not  thus  far 
yielded  results  equal  or  superior  to  ordinary  non-specific  antigens.  As 
compared  with  lipoidal  extracts,  they  have  generally  yielded  reactions 
that  are  much  weaker,  and  in  primary  and  secondary  syphilis  may  react 
entirely  negatively.  Much  is  yet  to  be  learned,  however,  of  bacterial 
antigens  in  general,  and  the  subject  must  be  regarded  as  still  in  the 
experimental  stage. 

It  may  be  said  to  be  well  proved  that  extract  antigens  in  the  syphilis 
reaction  are  not  biologically  specific,  and  need  not  be  extracts  of  syphilitic 
tissues.  An  antigen  cannot  give  reliable  or  satisfactory  results  unless  it  is 
carefully  titrated  and  its  properties  determined.  Antigens  may  serve  as  a 
frequent  source  of  error  when  the  complement-fixation  reactions  are  con- 
ducted by  those  possessing  insufficient  knowledge  of  their  good  and  bad 
properties.  The  test  for  the  syphilitic  reaction  should  not  be  undertaken 
by  any  one  not  competent  to  titrate  and  judge  of  the  qualities  of  the  antigen 
to  be  employed. 

Method  of  Diluting  Antigens.  As  a  general  rule,  all  organic  extracts 
must  be  diluted  with  normal  salt  solution  before  being  used. 

If  extracts  and  diluent  are  mixed  quickly,  the  emulsion  is  clear  or 
slightly  opalescent.    If  the  diluent  is  added  slowly  to  the  organic  extract, 
the  resulting  mixture  becomes  quite  turbid  and  milky.    As  shown  by 
Sachs  and  Roudoni,  the  antigenic  power  of  the  extract  is  more  marked 
with  the  turbid  than  when  the  clear  or  opalescent  emulsion  is  used.      , 
For  this  reason,  in  testing  for  the  syphilis  reaction  the  emulsion  of  organic  ^ 
extract  should  be  made  so  as  to  secure  the  maximum  amount  of  turbidity. 
The  required  amount  of  antigen  is  placed  in  a  test-tube,  and  the  salt  solution- 
is  added  slowly  with  a  pipet;  or  the  salt  solution  may  be  placed  in  a  tube  and 
the  extract  floated  on  it  and  gradually  mixed. 

Although  with  each  new  extract  it  may  be  necessary  to  titrate  with 
various  dilutions  before  one  that  is  satisfactory  is  reached,  experience 
has  shown  in  the  majority  of  instances  that  the  following  dilutions  are 
usually  correct: 

1.  Alcoholic  extract  of  syphilitic  liver:    1  part  with  9  parts  of  salt 
solution.     Extracts  of  German  manufacture  are  usually  diluted  six  or 
seven  times. 

2.  Cholesterinized  extracts  and  acetone-insoluble  lipoids  in  methyl 


452        THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

alcohol  require  higher  dilution,  as  1  part  of  extract  with  19  parts  of  salt 
solution. 

3.  Plain  alcoholic  extracts  of  human,  beef  and  guinea-pig  heart  like- 
wise require  high  dilution,  as,  for  example,  extract,  1   part  and  salt 
solution,  19  parts. 

4.  Alcoholic  extract  of  human  heart,  prepared  after  the  method  of 
Boas  (quick  method),  is  used  in  lower  dilution,  as  1  part  of  extract  with 
9  parts  of  salt  solution. 

5.  The   solution   of   lecithin   and   cholesterin   used   by   Browning, 
Cruickshank,  and  Mackenzie  is  diluted  with  seven  parts  of  salt  solution. 

6.  Aqueous  extracts  of  syphilitic  liver  and  extracts  of  pallida  culture 
are  used  undiluted,  or  may  require  dilution  with  four  parts  of  salt  solu- 
tion. 

J  Although  these  emulsions  mil  keep  for  a  few  days  if  placed  in  the  re- 
frigerator, it  is  advisable  to  make  up  only  the  amount  required  for  imme- 
diate use}  as  freshly  prepared  emulsions  are  better  than  older  ones. 

Method  of  Titrating  Antigens.     Three  values  are  to  be  determined: 

1.  The  anticomplementary  dose,  or  that  amount  of  antigen  that  in 
itself  is  capable  of  fixing  or  inactivating  the  complement. 

2.  The  hemolytic  dose,  or  that  amount  of  antigen  that  in  itself  is 
capable  of  lysing  red  blood-cells.    This  action  is  probably  due  to  the 
presence  of  certain  lipoids  and  alcohol. 

3.  The  antigenic  dose,  or  that  amount  of  antigen  that  serves  to 
absorb  or  fix  a  certain  and  constant  dose  of  complement  with  a  definite 
amount  of  syphilitic  serum. 

1.  Anticomplementary  Titration.  The  determination  of  the  anti- 
complementary  dose  is  probably  the  most  important,  for  if  an  extract 
were  used  in  an  amount  that  was  anticomplementary  or  capable  of 
fixing  complement  in  a  non-specific  manner,  all  the  tests  would  show 
false  positive  reactions,  regardless  of  whether  the  serum  was  from  a 
normal  or  from  a  luetic  person. 

After  determining  this  anticomplementary  dose,  in  conducting  the 
main  test  the  antigen  may  be  used  in  one-fourth  the  amount. 

All  tests  are  conducted  with  chemically  clean  and  preferably  sterile 
test-tubes  (12  cm.  by  13  mm.)  and  graduated  pipets.  Accuracy  in 
measurements  is  very  essential  in  performing  all  complement-fixation 
work. 

A  preliminary  titration  of  the  hemolysin  is  made,  with  the  complement 
and  corpuscle  suspension  to  be  used  in  titrating  the  antigen,  in  order  to 
determine  its  hemolytic  dose,  i.  e.,  to  adjust  the  hemolytic  system.  If, 


GENERAL   TECHNIC  453 

for  instance,  the  hemolytic  serum  is  known  to  have  a  titer  of  about 
1 : 2000  (see  p.  397),  a  stock  solution  is  made  by  diluting  1  c.c.  of  the  serum 
with  sterile  salt  solution  to  make  a  dilution  of  1:200.  In  a  series  of  six 
test-tubes  increasing  amounts  of  this  diluted  amboceptor  are  placed: 
0.05  c.c.,  0.1  c.c.,  0.15  c.c.,  0.2  c.c.,  0.3  c.c.,  and  0.4  c.c.;  to  each  tube  add 
1  c.c.  of  complement  Serum  diluted  1 : 20  (  =  0.05  c.c.  undiluted  serum) 
and  1  c.c.  of  a  2.5  per  cent,  suspension  of  sheep's  cells  and  sufficient  salt 
solution  to  bring  the  total  contents  up  to  3  or  4  c.c.  Each  tube  is  shaken 
gently  and  incubated  at  37°  C.  for  an  hour.  At  the  end  of  this  time  that 
amount  of  amboceptor  that  just  completely  hemolyses  the  corpuscles  is  taken 
ns  the  hemolytic  unit  (Fig.  106).  In  conducting  the  antigen  titrations 
twice  this  amount  is  used  as  the  dose. 

The  antigen  extract  is  diluted  1  : 10  by  placing  1  c.c.  in  a  test-tube  and 
slowly  adding  9  c.c.  of  normal  salt  solution. 

Increasing  amounts  of  this  emulsion  are  placed  in  a  series  of  test- 
tubes:  0.2,  0.4,  0.6,  0.8,  1,  1.2,  1.5,  and  2  c.c.  To  each  tube  are  now 
added  1  c.c.  of  the  diluted  complement  serum  ( =  0.05  c.c.  undiluted  se- 
rum) and  sufficient  normal  salt  solution  to  bring  the  total  volume  up  to  3 
c.c.  Shake  each  tube  gently  and  incubate  for  one  hour  at  37°  C.  Then 
add  to  each  tube  1  c.c.  of  the  corpuscle  suspension  and  a  dose  of  am- 
boceptor equal  to  2  units,  as  just  determined  by  previous  titration. 
Shake  gently  and  reincubate  for  another  hour  and  a  half,  when  a  prelim- 
inary reading  of  the  results  may  be  made.  That  amount  of  antigen  that 
shows  beginning  inhibition  of  hemolysis  is  regarded  as  the  anticomplemen- 
tary  unit.  The  final  readings  are  made  after  the  tubes  have  stood  over- 
night in  a  refrigerator  at  low  temperature  (Fig.  111). 

This  titration  may  also  be  made  in  the  presence  of  normal  serum, 
although  this  is  not  absolutely  necessary.  The  serum  must  be  perfectly 
fresh,  and  must  be  that  from  a  person  known  to  be  free  from  lues.  It  is 
inactivated  by  heating  to  55°  C.  for  half  an  hour,  and  0.2  c.c.  is  added 
to  each  tube.  Complement  and  salt  solution  are  now  added,  and  the 
titration  conducted  in  the  manner  just  described.  Normal  serum  may 
absorb  a  small  amount  of  complement  in  itself,  and  hence  a  titration 
conducted  with  serum  may  show  a  slightly  lower  anticomplementary 
dose. 

The  following  controls  are  included: 

1.  A  hemolytic  system  control,  containing  the  complement,  corpuscles, 
and  amboceptor  in  the  same  amounts  as  were  used  in  conducting  the 
titration.    This  control  should  show  complete  hemolysis. 

2.  A  serum  control,  which  is  the  same  as  the  hemolytic  system  control 
plus  0.2  c.c.  of  the  serum.     This  should  show  complete  hemolysis,  and 


454 


THE   TECHNIC   OF   COMPLEMENT-FIXATION   REACTIONS 


indicates  that  the  serum  was  not  anticomplementary.  This  control  test 
should  never  be  omitted. 

3.  A  corpuscle  control,  including  1  c.c.  of  the  corpuscles  in  salt  solu- 
tion. This  tube  should  show  no  hemolysis. 

The  following  table  gives  the  results  of  a  titration  with  an  alcoholic 
extract  of  syphilitic  liver  diluted  1:10  (see  Fig.  Ill): 

TABLE    13.— ANTICOMPLEMENTARY    TITRATION    OF    AN    ORGANIC 

EXTRACT 


SHEEP'S 

ANTIGEN 

COMPLE- 

NORMAL 

g  £ 

ANTI- 
SHEEP 

CORPUS- 

TUBE 

(1   10), 
C.c. 

MENT 
(1  :  20), 
C.c. 

SERUM, 
C.c. 

-P 

>    0) 

HEMOL- 

YSIN 

UNITS 

CLES 
2.5  PER 
CENT., 
C.c. 

RESULTS 

1... 

0.2 

1 

0.2 

|1 

2 

1 

Hemolysis 

2.  .  .  . 

0.4 

1 

0.2 

bC  o 

2 

1 

Hemolysis 

3.  ... 

0.6 

1 

0.2 

.s.s 

2 

1 

Hemolysis 

4..  .  . 

0.8 

1 

0.2 

,£573 

2 

1 

Hemolysis 

5.  .  .  . 

1.0 

1 

0.2 

3  3 

2 

1 

Hemolysis 

6.... 

1.2 

1 

0.2 

C  G 

2 

1 

Slight     inhibition    of 

.2  jjj 

hemolysis 

7.... 

1.5 

1 

0.2 

3  J5 

2 

1 

Marked  inhibition  of 

1  S 

hemolysis 

8.... 

2.0 

1 

0.2 

45  s 

2 

1 

Complete     inhibition 

M    ^ 

of  hemolysis 

9.... 

Control 

1 

— 

-4-i 

fl  T 

2 

1 

Hemolytic       control: 

10.  ... 

Control 

1 

0.2 

02    O 

"o  o 

2 

1 

complete  hemolysis 
Serum   control:   com- 

S3 

plete  hemolysis 

In  this  titration  tube  No.  6,  containing  1.2  c.c.  of  the  antigen  emulsion 
showed  beginning  inhibition  of  hemolysis  and  was  recorded  as  the  anti- 
complementary  dose. 

\l  2.  Hemolytic  Titration.  As  previously  mentioned,  organic  extracts 
are  capable  in  themselves  of  hemolyzing  red  cells;  this  is  due  to  the 
hemotoxic  action  of  lipoids  and  alcohol.  Extracts  of  organs  that  have 
undergone  advanced  autolysis  and  decomposition  are  very  likely  to  be 
hemolytic. 

Serum  exerts  an  inhibiting  influence  on  the  lytic  action  of  an  organic 
extract.  Hence  the  hemolytic  dose  of  an  extract  depends  largely  on 
whether  or  not  complement  serum  is  used  in  the  titration. 

When  an  organic  extract  is  titrated  in  the  presence  of  complement, 
the  hemolytic  dose  is  higher  than  the  anticomplementary  dose.  In  the 
foregoing  titration  3  c.c.  of  the  extract  emulsion  showed  beginning 
hemolysis,  and  when  4  c.c.  was  used,  hemolysis  was  complete.  These 
large  amounts  of  emulsion  give  the  tube  contents  quite  a  milky  appear- 
ance, but  close  inspection  shows  that  all  the  cells  are  broken  up. 


7 


• 


FlG.    111. — TlTRATION    OF   ANTIGEN   FOR   ANTICOMPLEMENTARY    UNIT. 


0.2 


FlG.    112. TlTRATION    OF   ANTIGEN   FOR   ANTIGENIC    UNIT. 

In  Fig.  Ill  the  tube  containing  1.5  c.c.  of  antigen  shows  slight  inhibition  of 
hemolysis,  and  this  amount  is  the  anticomplementary  unit. 

In  Fig.  112  the  tube  containing  0.15  c.c.  of  antigen  is  the  smallest  amount  pro- 
ducing complete  inhibition  of  hemolysis,  and  is  the  antigenic  unit. 


GENERAL   TECHNIC  455 

As  a  general  rule,  the  hemolytic  titration  is  not  absolutely  necessary. 
It  may  be  conducted  with  the  anticomplementary  titration  by  adding 
another  tube  or  two  to  the  foregoing  series,  with  higher  doses  of  extract; 
or  this  titration  may  be  conducted  separately,  and  without  complement 
and  hemolysin,  by  using  the  same  doses  of  antigen  with  1  c.c.  of  cor- 
puscle suspension  and  sufficient  salt  solution  to  bring  the  total  volume  in 
each  tube  up  to  3  or  4  c.c. 

3.  Antigenic  Titration. — As  previous^  stated,  this  titration  is  not 
absolutely  necessary,  as  one-fourth  the  anticomplementary  dose  of  an 
extract  may  be  used  in  the  main  test.  For  instance,  in  the  foregoing 
titration  0.3  or  0.4  c.c.  may  safely  be  used  in  making  the  test  for  the 
syphilitic  reaction.  Different  extracts  vary,  however,  in  their  anti- 
genie  value.  Some  may  be  highly  anticomplementary  and  have  a 
comparatively  low  antigenic  value;  purer  extracts,  such  as  acetone- 
insoluble  lipoids  or  cholesterinized  alcoholic  extracts  of  heart,  are 
largely  free  from  anticomplementary  action,  and  at  the  same  time 
possess  a  high  antigenic  value.  It  is  advisable,  therefore,  to  use  an  ^ 
antigen  whose  full  antigenic  as  well  as  anticomplementary  doses  are 
known,  for,  while  it  is  necessary  to  use  sufficient  antigen,  it  is  not  advisable 
to  use  a  larger  amount  than  is  necessary. 

For  this  titration  all  antigens  except  alcoholic  extracts  of  syphilitic 
liver,  should  be  diluted  1 : 20  with  normal  salt  solution.  Usually  the 
antigenic  unit  is  so  much  lower  than  the  anticomplementary  unit  that 
it  is  best  determined  with  a  more  dilute  antigen. 

The  titration  is  conducted  in  a  manner  similar  to  the  anticomple- 
mentary titration,  except  that  0.2  c.c.  of  fresh  and  inactivated  serum 
from  a  known  and  untreated  syphilitic  person  is  added  to  each  tube. 
Increasing  doses  of  antigen,  patient's  serum,  and  complement  are 
mixed,  shaken,  and  incubated  for  one  hour.  Two  units  of  hemolysin 
and  corpuscles  are  then  added,  the  tubes  shaken  and  incubated  for 
another  hour,  after  which  the  preliminary  reading  is  made.  The  final 
reading  is  taken  after  the  tubes  have  been  placed  over  night  in  a  refriger- 
ator at  low  temperature.  That  amount  of  antigen  that  shows  just  complete 
inhibition  of  hemolysis  is  taken  as  the  antigenic  unit  (Fig.  112).  In  con- 
ducting the  syphilis  reaction  two  to  four  times  this  unit  is  used,  providing 
that  these  amounts  are  at  least  four  or  five  times  less  than  the  anticomple- 
mentary dose.  This  larger  antigenic  dose  is  advisable,  because  the  exact 
unit  may  not  be  sufficient  with  serums  containing  but  small  amounts 
of  syphilis  antibody  such  as  those  of  treated  or  long-standing  cases  of 
lues. 


456         THE    TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 


The  following  table  illustrates  this  titration  with  the  same  alco- 
holic extract  of  syphilitic  liver  (see  Fig.  112): 

TABLE  14.— ANTIGENIC  TITRATION  OF  AN  ORGANIC  EXTRACT 


TUBE 

ANTI- 
GEN 
1:10, 
C.c. 

SYPHI- 
LITIC 
SERUM 
(INACTIVE) 
C.c. 

COM- 
PLE- 
MENT 
1:20, 
C.c. 

o>    . 
,5  o 

>    0 

~  a 

3T3 

ANTI- 
SHEEP 
HEM- 

OLYSIN, 

DOSES 

SHEEP 
COR- 
PUSCLES 
2.5 
PER 
CENT., 
C.c. 

,  preliminary 

RESULTS 

1  

0.05 

0.2 

1 

-Si 

2 

1 

i 

Slight  inhibition 

2  
3  
4.. 

0.1 
0.15 
0.2 

0.2 
0.2 
02 

1 
1 
1 

olution  to  bring  1 
shaken  and  incu 

2 
2 

2 

1 
1 
1 

d  incubated  one  h 
reading. 

of  hemolysis 
Marked     inhibi- 
tion of  hemoly- 
sis 
Complete     inhi- 
bition of  hemol- 
ysis; unit. 
No  hemolysis 

5  
6 

0.25 
0.3 

0.2 
02 

1 
1 

si 

*c3   3 

2 

2 

1 
1 

d 

No  hemolysis 
No  hemolysis 

7  

0 

Control 
Control 

0.2 
0 

1 
1 

2 

0 

1 
1 

1 

i 

Serum      control: 
Hemolysis 
Hemolytic    con- 

^3 

J 

trol:     Hemoly- 
sis 

In  this  instance  0.15  c.c.  of  the  emulsion  represents  the  antigenic 
unit.  In  performing  the  Wassermann  reaction  0.3  or  0.4  c.c.  was  used, 
and  these  amounts  were  about  one-fourth  the  anticomplementary  dose. 

It  is  not  unusual  to  find  cholesterinized  alcoholic  extract  and 
acetone-insoluble  lipoids  perfectly  antigenic  in  0.05  c.c.  of  a  1:20 
dilution,  and  not  anticomplementary  under  1  or  2  c.c.  of  a  1:10  dilu- 
tion. In  these  instances  four  times  the  antigenic  dose,  or  0.2  c.c. 
can  be  used,  and  yet  this  amount  is  at  least  10  times  smaller  than  the 
anticomplementary  dose — a  condition  of  affairs  that  constitutes  a  safe 
and  desirable,  antigen. 

Each  new  antigen  should  be  tested  with  a  number  of  serums  and 
controlled  by  an  older  antigen  of  known  value  before  being  finally 
accepted  as  satisfactory. 

Antigen  containers  should  be  well  stoppered  and  kept  in  the  refrig- 
erator. Deterioration  may  set  in  suddenly,  and  they  should,  therefore, 
be  retit rated  every  few  weeks. 

VARIOUS  METHODS  FOR  CONDUCTING  THE  SYPHILIS  REACTION 

First  Method;  the  Original  Wassermann  Reaction. — The  simplest 
technic,  and  the  one  best  adapted  for  inexperienced  workers,  is  the 
original  Wassermann  reaction,  performed  with  alcoholic  instead  of 


METHODS    FOR    CONDUCTING    THE    SYPHILIS    REACTION  457 

aqueous  extracts  of  syphilitic  liver  as  antigen.  It  is  true  that  this  method 
is  not  an  exact  quantitative  reaction,  and  that  it  is  probably  less  delicate 
than  some  of  the  modified  methods,  but  its  advantages  are  that  it  is 
easy  of  manipulation,  is  readily  learned,  and  is  especially  recommended 
for  persons  who  perform  these  tests  at  irregular  intervals,  as  false  positive 
reactions  are  less  likely  to  occur  than  when  the  more  delicate  methods 
are  used. 

Second  Method ;  the  Wassermann  Reaction  with  Multiple  Antigens. 
— In  this  method  the  technic  is  essentially  the  same  as  in  the  first 
method,  except  that  three  different  antigens  are  used  instead  of  one, 
namely,  cholesterinized  extract  of  normal  heart,  alcoholic  extract  of 
syphilitic  liver,  and  acetone-insoluble  lipoids.  This  method  has  three 
advantages:  (1)  It  permits  the  use  of  a  cholesterinized  extract  under 
conditions  where  any  tendency  to  non-specific  fixation  is  to  be  controlled ; 
(2)  an  antigen  may  at  any  time  suddenly  become  anticomplementary 
and  yield  false  results,  whereas  by  this  method  the  source  of  error  is 
detected  and  may  be  avoided,  since  it  is  not  dependent  upon  any  one 
extract;  (3)  an  extensive  study  of  the  comparative  values  of  antigens 
has  led  to  the  distinct  impression  that  the  lipodophilic  antibody  in 
different  syphilitic  serums  frequently  shows  a  special  affinity  for  the 
lipoids  in  a  certain  plain  antigen  more  than  it  does  for  those  in 
another  antigen;  in  fact,  I  have  not  infrequently  found  that,  with 
weakly  positive  serums,  if  one  antigen  had  been  emploj^ed,  a  false 
negative  report  would  have  been  rendered,  the  true  reaction  being 
given  by  the  other  two  antigens.  These  results  could  not  be  as- 
cribed to  faulty  antigen,  for  with  other  weakly  positive  serums  the  ex- 
tract would  be  found  to  react  satisfactorily. 

As  previously  mentioned,  cholesterinized  alcoholic  extracts  are 
very  sensitive,  so  that  from  this  standpoint  additional  antigens  would 
appear  to  be  superfluous.  This  very  property,  however,  in  my 
opinion,  renders  it  advisable  to  control  them  with  less  sensitive  ex- 
tracts. In  this  way  all  the  advantages  of  a  very  sensitive  antigen 
may  be  secured,  and  the  disadvantages  avoided  until  more  extended 
use  demonstrates  whether  or  not  it  is  entirely  safe  to  use  these  extracts 
alone. 

With  strongly  reacting  serums  all  antigens  possess  equal  antigenic 
power.  With  the  serums  of  long-standing  or  treated  cases  of  syphilis 
the  cholesterinized  extracts  may  react  strongly  positive,  whereas  with 
the  aqueous  and  the  alcoholic  extracts  the  reactions  are  weakly 
positive,  or  negative  with  one  and  positive  with  the  other.  In  cases 
of  syphilis  that  have  received  considerable  treatment  the  reaction 


458        THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

may  be  negative  at  first  with  the  aqueous  and  the  alcoholic  extracts, 
and  as  treatment  is  continued  it  may  finally  be  negative  with  the 
cholesterinized  extracts.  In  a  certain  percentage  of  cases,  especially 
those  of  old  infections  of  the  central  nervous  system,  the  reaction  is 
positive  with  the  cholesterinized  extract  and  negative  with  the  other 
extracts;  strong  reactions  of  this  character  usually  indicate  syphilitic 
infection.  Occasionally  a  weak  (10  per  cent,  or  less,  inhibition  of 
hemolysis)  reaction  may  be  had  with  the  serum  of  a  person  who  denies 
syphilis. 

Third  Method;  the  Wassermann  Reaction  with  Varying  Amounts 
of  Patient's  Serum. — One  disadvantage  of  the  regular  Wassermann 
technic  is  that  it  may  not  readily  show  improvement  of  the  patient 
while  the  treatment  is  going  on.  For  instance,  if  complete  fixation 
of  complement  occurs  with  0.2  c.c.  of  serum,  one  does  not  know 
whether  this  is  the  smallest  fixing  dose,  or  whether  there  might  be  a 
fixation  even  with  much  smaller  quantities  of  serum.  This  is  very 
important  in  examining  cases  during  the  course  of  the  treatment,  as 
otherwise  improvement  in  the  condition  may  be  overlooked.  Just 
as  soon,  however,  as  the  reaction  with  0.2  c.c.  of  serum  is  a  degree 
less  than  absolutely  positive,  then  the  various  steps,  down  to  com- 
plete negative  reactions,  are  readily  observed  and  recorded  by  the 
usual  Wassermann  technic.  This  disadvantage  may  be  overcome 
by  using  at  least  seven  different  doses  of  serum:  0.01,  0.02,  0.04,  0.06, 
0.08,  0.1,  0.2  c.c.  In  this  way  a  means  is  afforded  for  judging  of  the 
strength  of  the  reaction,  and  the  effect  of  antisj^philitic  treatment  is 
readily  observed. 

Fourth  Method;  the  Wassermann  Reaction  with  Varying  Amounts 
of  Complement. — It  has  previously  been  pointed  out  that  the  syphilis 
reaction  is  dependent  upon  the  fact  that  while  hemolytic  complement 
may  be  rendered  inactive  or  fixed  by  serum  alone  and  organic  extract 
alone,  it  is  characteristic  of  syphilis  that  a  mixutre  of  serum  and  extract 
will  absorb  or  fix  more  complement  than  the  sum  of  the  amounts  absorb- 
ed by  these  two  substances  alone.  In  the  foregoing  methods  no  attempt 
has  been  made  to  measure  the  amount  of  complement  absorbed  by  serum 
and  antigen  alone,  but  sufficient  complement  has  been  furnished  to  allow 
for  this  non-specific  fixation,  and  we  are  content  to  show  that  the  serum 
and  antigen  alone  do  not  absorb  enough  complement  to  interfere  with 
hemolysis,  so  that  any  inhibition  of  hemolysis  may  be  interpreted  as 
specific  complement  fixation. 

Browning  and  Mackenzie  and  Thomsen  have  devised  a  technic  where- 
by it  is  possible  to  estimate  the  actual  amounts  of  complement  absorbed, 


METHODS  FOK  CONDUCTING  THE  SYPHILIS  REACTION          459 

first,  by  the  serum  and  antigen  alone,  and  second  by  these  two  sub- 
stances combined.  The  complement  absorbed  is  measured  in  terms 
of  hemolytic  doses.  This  method  consumes  a  little  more  time  and 
more  of  the  various  reagents  is  required.  It  is,  nevertheless,  the 
best  quantitative  method  we  have,  and  shows  exactly  the  degree  of 
complement  fixation  in  each  case. 

In  conducting  any  complement-fixation  test  the  following  are 
essential  factors  if  success  is  to  be  achieved:  (1)  Reliable  reagents, 
particularly  a  good  antigen  must  be  had,  for  no  matter  how  much 
care  is  exercised,  good  results  cannot  be  secured  with  indifferent 
reagents;  (2)  the  observer  must  possess  a  thorough  working  under- 
standing of  the  underlying  principles  and  particularly  of  the  quanti- 
tative relations  of  the  various  reagents;  (3)  there  must  be  an  accurate 
adjustment  of  the  hemolytic  system;  (4)  he  must  have  a  careful, 
painstaking  and  accurate  habit  of  pipeting  small  amounts.  Accuracy 
should  never  be  sacrificed  for  speed,  as  the  latter  is  properly  acquired 
only  with  experience. 

TECHNIC  OF  THE  FIRST  METHOD 
THE  ORIGINAL  WASSERMANN  REACTION 

This  is  the  original  Wassermanri  reaction,  except  that  an  alcoholic, 
instead  of  an  aqueous  extract  of  syphilitic  liver,  is  used  as  antigen. 
This  is  the  simplest  of  all  technics,  and,  when  properly  performed,  con- 
stitutes, in  the  final  analysis,  a  reliable  test  and  one  especially  adapted 
for  those  not  constantly  engaged  in  this  work. 

1.  Complement. — Fresh  clear  serum  (not  over  twenty-four  hours 
old)  of  a  healthy  guinea-pig.    Dilute  1  :  10  by  adding  9  c.c.  of  sterile 
normal  saline  solution  to  each  1  c.c.  of  serum.    Dose,  1  c.c.  (=  0.1  c.c. 
of  undiluted. serum). 

2.  Corpuscles. — Sheep's  blood  washed  three  times  and  diluted  to 
make  a  5  per  cent,  suspension.     For  example,  1  c.c.  of  corpuscles  in 
19  c.c.  of  salt  solution  makes  up  sufficient  for  a  number  of  tests. 

3.  Hemolytic   Amboceptor. — Serum   of  a   rabbit  immunized   with 
washed  sheep's  corpuscles.    As  stated  elsewhere,  this  serum  is  heated 
to  55°  C.  for  half  an  hour,  and  an  equal  part  of  chemically  pure  glycerin 
is  added.    Mix  well  and  preserve  in  sterile  1  c.c.  ampules.    Each  am- 
pule will,  therefore,  contain  0.5  c.c.  of  serum.     One  stock  dilution  is 
prepared  in  such   manner  that  about  0.2  c.c.  represents  one  hemo- 
lytic dose.     One  may  otherwise  prepare  a  whole  series  of  flasks  with 
various  dilutions,  and  in  making  a  titration  to  use  1  c.c.  of  each  di- 


460  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

lution;  I  have  found  it  much  more  accurate,  simple,  and  economical, 
however,  to  prepare  one  stock  dilution,  which  is  titrated  with  each 
complement  and  corpuscle  suspension  before  each  day's  work.  For 
example,  if  a  serum  is  known  to  have  a  titer  of  1  :  2000,  an  ampule 
(0.5  c.c.  of  serum)  is  diluted  with  200  c.c.  of  salt  solution;  this  gives  a 
dilution  of  serum  approximately  1:400,  of  which  0.2  represents  one 
hemolytic  dose.  The  titration  must  be  repeated  each  time  to  make 
sure  of  this,  because  the  complement  of  different  pigs  may  vary  in 
activity,  and  the  chief  object  is  to  adjust  the  amboceptor  and  com- 
plement to  each  other. 

Titration  of  Amboceptor. — Into  a  series  of  six  test-tubes  place  in- 
creasing amounts  of  the  amboceptor  dilution:  0.05  c.c.,  0.1  c.c.,  0.15 
c.c.,  0.2  c.c.,  0.25  c.c.,  and  0.3  c.c.  Add  1  c.c.  of  complement  (1  : 10) 
and  1  c.c.  of  corpuscle  suspension  to  each  tube,  and  sufficient  salt 
solution  to  make  the  total  volume  in  each  tube  about  4  c.c.  Shake 
gently  and  incubate  for  one  hour  at  37°  C.  At  the  end  of  this  time 
the  tube  showing  just  complete  hemolysis  contains  one  hemolytic 
dose,  or  unit  of  amboceptor.  In  the  tests  double  this  amount,  or  two 
units,  is  used. 

The  amboceptor  titration  is  very  important.  Under  no  cir- 
cumstances should  the  same  dose  be  used  day  after  day  without 
titration,  because  the  complement  of  different  guinea-pigs  may  vary 
in  its  activity,  and  these  variations  would  be  detected  and  would  be 
adjusted  in  this  titration.  For  example,  with  a  weaker  complement 
the  dose  of  amboceptor  required  to  effect  complete  hemolysis  becomes 
higher;  each  new  corpuscle  suspension  may  also  vary  slightly  in  the 
actual  number  of  cells  contained  in  1  c.c.,  but  this  makes  no  difference 
when  each  suspension  is  titrated  with  the  complement  and  ambo- 
ceptor to  be  used  in  the  day's  work.  This  titration  is  set  up  first, 
and  while  it  is  in  the  incubator,  the  main  tests  are  arranged. 

4.  Antigen. — Alcoholic  extract  of  syphilitic  liver  or  acetone- 
insoluble  lipoids  of  proved  value  may  be  used.  It  is  well  to  estimate 
just  how  much  antigen  will  be  required  for  the  tests  on  hand,  so  that 
no  waste  will  occur,  as  fresh  emulsions  are  better  than  old  ones  carried 
over  from  day  to  day.  The  dose  should  be  at  least  double  the  titrated 
antigenic  unit,  or  one-fourth  of  the  anticomplementary  dose.  For 
instance,  if  an  alcoholic  extract  of  syphilitic  liver  diluted  1  :  10  is 
found  on  titration  to  be  perfectly  antigenic  in  doses  of  0.2  c.c.,  and 
not  anticomplementary  in  amounts  under  2  c.c.,  then  0.4  c.c.  maybe 
used  in  making  the  tests,  as  this  amount  is  still  about  five  times  less 


METHODS  FOR  CONDUCTING  THE  SYPHILIS  REACTION          461 

than  the  anticomplementary  dose,  and  well  within  the  range  of  safety 
against  non-specific  complement  fixation.  If  10  tests  are  to  be  made, 
then  at  least  4.4  c.c.  of  diluted  antigen  are  required,  including  sufficient 
for  the  antigen  control,  or  in  round  numbers,  0.5  c.c.  of  antigen  plus  4.5 
c.c.  of  salt  solution  in  order  to  secure  the  maximum  turbidity  slowly  added. 

5.  Serum. — Serum  should  be  fresh  and  clear  and  heated  in  a  water- 
bath  to  55°  C.  for  half  an  hour  before  using.     The  temperature  should 
not  go  above  56°  C.  nor  below  55°  C.     The  complement  in  perfectly 
fresh  serum  is  usually  inactivated  at  this  temperature  within  fifteen 
minutes,  but  it  is  better  to  adopt  the  period  of  one-half  hour  as  a 
routine,  especially  in  removing  heat-sensitive  anticomplementary  sub-  > 
stances  that  develop  in  serums  more  than  a  day  old.    Dose,  0.2  c.c. 

6.  Cerebrospinal  Fluid. — This  should  be  fresh  and  free  from  blood. 
It  is  used  unheated,  as  spinal  fluid  contains  little  or  no  hemolytic  <- 
complement.    The  dose  should  be  at  least  four  times  that  of  the  serum, 
or  0.8  c.c. 

The  Test. — A  front  and  a  rear  tube  for  each  serum  are  placed  in  a 
rack.  Each  tube  is  marked  plainly  with  the  patient's  name  or  in- 
itials, and  in  addition  the  front  tube  is  marked  with  the  number  of 
the  antigen  or  with  the  letter  "A, "  or  the  word  " antigen"  is  written 
on  it,  the  rear  tube  being  marked  " control"  (serum  control).  The 
necessity  for  carefully  marking  each  tube  is  nowhere  more  important 
than  in  conducting  Wassermann  reactions  with  a  number  of  serums, 
as  the  slightest  error  or  lapse  of  memory  may  result  in  confusion  and 
prove  to  be  quite  a  serious  matter. 

In  each  series  of  reactions  -the  serum  from  a  known  case  of  syphilis 
that  has  given  a  positive  reaction  and  the  serum  of  a  known  non- 
syphilitic  person  are  included  as  positive  and  negative  controls  re- 
spectively. 

Into  each  front  tube  the  proper  dose  of  antigen  is  placed;  to  the 
front  and  rear  tubes  0.2  c.c.  of  the  patient's  serum  is  added.  To  all 
tubes  1  c.c.  of  the  complement  (1  :  10)  and  sufficient  normal  salt  solu- 
tion are  then  added  to  bring  the  total  volume  in  each  to  about  3  c.c. 

The  rear  tube  of  each  set  is  the  serum  control;  the  positive  and 
negative  serums  are  treated  in  just  the  same  manner  as  the  patient's 
serum.  In  addition  to  these  there  are  three  other  important  controls 
that  should  not  be  omitted: 

1.  The  antigen  control:    Dose  of  antigen  plus  1  c.c.  of  complement 
(1  :  10)  and  a  sufficient  quantity  of  salt  solution. 

2.  Hemolytic  system  control:    1  c.c.  of  complement  (1  :  10)  and  2  c.c. 
of  salt  solution. 


462 


THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 


3.  Corpuscle  control:  1  c.c.  of  corpuscle  suspension  plus  3  c.c.  of 
salt  solution. 

Each  tube  is  gently  shaken  and  incubated  at  37°  C.  for  an  hour, 
when  two  hemolytic  doses  of  amboceptor  and  1  c.c.  of  corpuscle  sus- 
pension (5  per  cent.)  are  added  to  each  tube  except  the  corpuscle  con- 
trol. Tubes  are  shaken  and  reincubated  for  an  hour  or  an  hour  and 
a  half,  depending  upon  the  hemolysis  of  the  serum  controls,  after 
which  a  preliminary  reading  is  made  and  recorded.  With  partially 
positive  reactions  the  tubes  may  be  centrifuged  in  order  to  read  the 
relative  amounts  of  hemolysis,  and  the  final  reading  made  at  once,  or 
the  tubes  may  be  placed  in  the  refrigerator  (just  above  freezing- 
point)  and  the  final  readings  made  the  next  morning. 

TABLE  15.— SCHEME  FOR  CONDUCTING  A  WASSERMANN  REACTION 
(FIRST  METHOD)  (SEE  FIG.  113) 


UNKNOWN  SERUM, 
MR.  B. 

UNKNOWN  CERE- 
BROSPINAL FLUID, 
MR.  C. 

KNOWN  POSITIVE 
SYPHILITIC 
SERUM 

KNOWN  NEGATIVE 
NORMAL  SERUM 

CONTROLS 

2. 
Serum,  0.2  c.c. 

+ 
Complement  (1 
c.c.  of  1  :  10)  + 
Salt     solution) 
(q.  s.  3  c.c.) 

4. 
Cerebrospinal 
fluid,  0.8  c.c.  + 
Complement  (1 
c.c.  of  1  :  10)  + 
Salt      solution 
(q.  s.  3  c.c.) 

6. 
Serum,  0.2  c.c. 
+ 
Complement  (1 
c.c.  of  1  :  10)  + 
Salt      solution 
(q.  s.  3  c.c.) 

8. 
iSerum,  0.2  c.c. 
+ 
Complement  (1 
c.c.  of  1:20)  + 
Salt  solution 
(q.  s.  3  c.c.) 

10. 

Antigen  control  : 
Antigen,        0.4 
c.c.  + 
Complement  (1 
c.c.  of  1:10)  + 
Salt       solution 
(q.  s.  3  c.c.) 

1. 
Antigen,  0.4  c.c. 
+ 
Serum,  0.2  c.c. 
+ 
Complement  (1 
c.c.  of  1:10)  + 
Salt       solution 
L  (q.  s.  3  c.c.) 

3. 
Antigen,       0.4 

C.C.+ 

Cerebrospinal 
fluid,  0.8  c.c. 
+ 
Complement  (1 
c.c.  of  1:10)  + 
Salt      solution 
(q.  s.  3  c.c.) 

5. 
Antigen,       0.4 

C.C.+ 

Serum,  0.2  c.c. 
+ 
Complement  (1 
c.c.  of  1:10)  + 
Salt      solution 
(q.  s.  3  c.c.) 

7. 
Antigen,       0.4 

C.C.+ 

Serum,  0.2  c.c. 
Complement  (1 
c.c.  of  1:20)  + 
Salt      solution 
(q.  s.  3  c.c.) 

9. 
Hemolytic  con- 
trol. 
Complement  (1 
c.c.  of  1:10)  + 
Salt       solution 
(q.  s.  3  c.c.) 

Tubes  are  shaken  gently  and  incubated  at  37°  C.  for  an  hour,  after  which  two 
hemolytic  doses  of  amboceptor  and  1  c.c.  of  corpuscle  suspension  are  added  to  each. 
They  are  then  gently  shaken  and  reincubated  for  an  hour  or  an  hour  and  a  half,  after 
which  a  preliminary  reading  is  made. 

All  the  tubes  in  the  rear  row  (upper  row  in  table)  (serum  controls),  the  antigen 
and  hemolytic  system  controls,  and  the  front  tube  with  the  negative  normal  serum, 
are  completely  hemolyzed.  The  front  tubes  with  the  unknown  serum  and  Cerebro- 
spinal fluid  and  the  positive  serum  control  show  inhibition  of  hemolysis  or  positive 
reactions. 

This  scheme  illustrates  the  technic  employed  with  an  unknown 
serum  and  Cerebrospinal  fluid.  The  proper  dose  of  diluted  antigen  is 
taken  as  0.4  c.c.,  and  two  doses  of  hemolytic  amboceptor  determined 
by  titration  as  equivalent  to  0.4  c.c.  of  the  stock  dilution. 


M 


ill 


I  I 


FIG.  113. — WASSERMANN  REACTION  (FIRST  METHOD). 


H 


FIG.  114. — READING  THE  WASSERMANN  REACTION. 


METHODS    FOR    CONDUCTING    THE    SYPHILIS    REACTION  463 

READING  AND  RECORDING  THE  WASSERMANN  REACTION 

1.  The  hemolytic  system  control  is  inspected  first.     It  should  show 
complete  hemolysis,  indicating  that  the  complement  and  amboceptor 
were  active  and  have  been  used  in  sufficient  amounts.     If  a  few  cor- 
puscles are  found  in  the  bottom  of  the  tube,  some  error  in  pipeting 
has  probably  occurred,  too  many  corpuscles  or  too  little  complement 
or  amboceptor  having  been  introduced. 

2.  The  corpuscle  control  should  show  no  hemolysis,  indicating  that 
the  solution  is  isotonic  and  that  the  corpuscles  are  not  unduly  fragile. 

3.  The  antigen  control  should  show  complete  hemolysis,  indicat- 
ing that  the  dose  used  was  not  anticomplementary.     If  this  tube 
shows  incomplete  hemolysis,  due  to  the  anticomplementary  action 
of  the  antigen,  all  the  front  two  tubes  will  also  show  some  inhibition 
of  hemolysis,  due  to  this  non-specific  complement  fixation,  and  it  is 
necessary  to  repeat  the  tests  with  another  extract. 

4.  The  rear  tubes  of  all  serums  should  be  completely  hemolyzed, 
indicating  that  the  serums  were  practically  free  from  anticomple- 
mentary action  as  previously  stated,  most  antigens  and  serums  are 
usually  very  slightly  anticomplementary  if  small  amounts  of  com- 
plement are  used  with  a  close  single  unit  of  amboceptor,  but  in  this 
technic  the  complement  and  two  units  of  amboceptor  are  sufficient, 
under  ordinary  circumstances,  to  offset  this  influence.     If,  however, 
a  serum  is  more  than  normally  anticomplementary,  the  rear  tube 
will  show  some  inhibition  of  hemolysis,  and,  of  course,  in  the  front 
tube  a  similar  inhibition,  and  probably  to  a  greater  degree,  will  be 
seen.     If  the  serum   is   very   slightly   anticomplementary   and   the 
front  tube  shows  complete  inhibition  of  hemolysis,  the  reaction  is  in 
all  probability  positive.     If  the  rear  tube,  however,  shows  marked 
inhibition  of  hemolysis,  indicating  that  it  is  highly  anticomplementary, 
the  result  cannot  be  determined,  but  a  retest  with  fresh  serum  must     / 
be  made.     This  indicates  the  great  importance  of  the  "serum  control,"    * 
and  it  may  be  stated  that  a  test  should  never  be  made  without  it. 

5.  The  front  tube  containing  the  known  syphilitic  serum  should  ^ 
show  inhibition  of  hemolysis,  indicating  that  the  extract  possesses 
antigenic  properties. 

As  the  complement  is  "fixed"  by  the  syphilis  antibody  and  ex- 
tract, hemolysis  could  not  occur  when  the  corpuscles  and  amboceptor 
were  added.  If  a  portion  of  the  complement  is  fixed  by  antibody  and 
extract,  then  the  unfixed  portion  will  hemolyze  some  of  the  corpuscles, 
the  reaction  being  moderately  positive,  slightly  positive,  etc.,  depend- 


464  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

ing  upon  the  degree  of  hemolysis  that  takes  place.  This  illustrates 
the  importance  of  observing  exactness  in  pipeting,  and  the  great 
influence  of  quantitative  factors  in  testing  for  the  Wassermann  reac- 
tion, for  if  an  excess  of  complement  is  used,  there  may  be  sufficient 
for  all  the  syphilis  antibody,  and  enough  unbound  complement  to 
hemolyze  all  the  corpuscles.  In  this  manner  a  false  negative  reaction 
will  result.  Corpuscles  and  sufficient  hemolytic  amboceptor  are 
added  merely  in  order  to  test  for  any  free  complement.  Under  proper 
conditions  a  total  lack  of  hemolysis  indicates  that  there  is  no  free 
complement,  but  that  it  has  been  fixed  by  syphilis  antibody  and 
extract,  constituting  a  positive  reaction  (+  -f-  +  +).  Complete 
hemolysis  indicates  that  complement  was  not  bound  and  that  syphilis 
antibody  was,  therefore,  absent  from  the  fluid  tested  —  a  negative 
reaction  (  —  ).  Partial  hemolysis  indicates  that  a  portion  of  the 
complement  has  been  fixed  by  smaller  amounts  of  syphilis  antibody 
and  of  the  extract,  yielding  partially  positive  reactions  (+  +  +; 

+  +;  +;  *). 

6.  The  front  tube  containing  the  known  normal  serum  should  show 
complete  hemolysis  because,  in  the  absence  of  syphilis  antibody,  the 
complement  remains  free  to  hemolyze  the  corpuscles  with  the  hemo- 
lytic amboceptor. 

7.  Various  methods  have  been  proposed  for  recording  the  results 
of  hemolytic  tests.     The  following  scheme,  after  Citron,  is    widely 
used  (Fig.  114): 


=  complete   inhibition   of    hemolysis  =  strongly  positive. 
+  +  +  =  75  per  cent,  inhibition  of  hemolysis  =  -moderately  positive. 
+  +  =  50  per  cent,  inhibition  of  hemolysis  =  weakly  positive. 
-h  =  25  per  cent,  inhibition  of  hemolysis  =  very  weakly  positive. 
=*=  =  less  than  25  per  cent,  inhibition  of  hemolysis  =  delayed 

hemolysis  or  doubtful  reaction. 
—  =  complete  hemolysis  =  negative  reaction. 

Under  the  third  method  a  scale  is  given  that  is  easily  prepared 
for  making  these  readings.  However,  after  some  experience  they  are 
readily  made,  and  at  first  should  be  attempted  only  after  the  non- 
hemolyzed  corpuscles  have  been  centrifuged  or  allowed  to  settle  to  the 
bottom  of  the  tube.  As  stated  elsewhere,  this  method  is  not  an 
accurate  measure  of  the  amount  of  syphilis  antibody,  but  constitutes 
a  relative  and  convenient  gage  of  value  within  certain  limits.  In 
reporting  reactions  to  the  clinician,  the  plus  signs  should  not  be  used, 
or  if  used,  should  be  interpreted  by  the  terms  "strongly  positive," 
"weakly  positive,  "  etc. 


METHODS    FOR    CONDUCTING    THE    SYPHILIS    REACTION  465 

TECHNIC  OF  THE  SECOND  METHOD 
THE  WASSERMANN  REACTION  WITH  MULTIPLE  ANTIGENS 

Practically  the  same  technic  is  used  in  this  as  in  the  first  method, 
except  that  three  different  antigens,  instead  of  one,  are  used  with  each 
serum,  for  the  reasons  previously  stated;  for  economy  the  amounts  of 
each  reagent  are  just  one-half  those  originally  employed. 

This  method  is  to  be  strongly  recommended,  as  it  is  simple,  ac- 
curate, and  reliable.  Although  a  little  more  work  is  demanded  and 
a  larger  quantity  of  the  various  reagents  is  required,  the  results 
warrant  the  expenditure  of  a  little  more  labor,  and  the  second  ob- 
jection is  readily  overcome  by  using  half  the  quantities  prescribed 
in  the  original  Wassermann  technic,  as  given  in  the  first  method. 

1.  I  generally  use  the  following  three  antigens:    (1)    A  cholesterin- 
ized  alcoholic  extract  of  human  heart;    (2)  alcoholic  extract  of  syphil- 
itic liver;   (3)  acetone-insoluble  lipoids. 

As  previously  stated,  these  extracts  are  used  in  amounts  equal  to 
from  two  to  four  times  their  titrated  antigenic  unit,  providing  these 
doses  are  at  least  four  times  smaller  than  the  anticomplementary 
units.  The  amount  of  each  antigen  required  for  the  work  at  hand 
is  calculated,  placed  in  test-tubes,  and  slowly  diluted  with  the 
requisite  amount  of  salt  solution  to  secure  maximum  turbidity  of  the 
emulsions. 

2.  The  complement  is  diluted  1  :  20  and  is  used  in  doses  of  1  c.c.; 
sheep's  corpuscles  are  made  up  into  a  2.5  per  cent,  suspension,  and 
used  in 'doses  of  1  c.c.;   antisheep  amboceptor  is  titrated  as  in  the  first 
method,  and  used  in  doses  equal  to  2  units;    serums  are  heated  to 
55°  C.  for  half  an  hour,  and  used  in  doses  of  0.1  to  0.2  c.c.;  cerebro- 
spinal  fluid  is  used  unheated  in  amounts  of  0.8  c.c.    With  fresh  sera  I      / 
use  routinely  0.2  c.c.  as  the  dose;  the  0.1  c.c.  dose  is  used  in  case  the  v 
amount  of  serum  at  hand  is  insufficient  for  the  0.2  c.c.  dose,  or  in  case 
the  serum  is  suspected  as  containing  thermostabile  anticomplementary 
substances. 

The  complement  may  be  titrated  instead  of  the  hemolysin  (technic 
on  page  398)  and  used  in  a  dose  equal  to  2  units.  A  large  series  of 
comparative  tests  with  both  methods  and  the  same  sera  have  yielded 
almost  identical  results. 

The  Test. — For  each  serum  four  test-tubes  are  arranged  in  a  row 

and  marked  with  the  patient's  name  or  initials.     The  first  tube  is 

marked  "C.  H.,"  and  receives  the  cholesterinized  heart  extract;   the 

second  tube  is  marked  "S"  for  the  alcoholic  extract  of  syphilitic 

30 


466  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

liver;  the  third  is  marked  "A"  for  acetone-insoluble  lipoids,  and  the 
fourth  is  not  marked  at  all  or  simply  marked  with  the  letters  aS.  C." 
(serum  control). 

To  each  of  the  four  tubes  0.1  or  0.2  c.c.  of  the  patient's  serum  is 
added,  or  0.8  c.c.  of  cerebrospinal  fluid. 

To  each  tube  1  c.c.  of  the  diluted  complement  (1  :  20)  and  suffi- 
cient salt  solution  to  bring  the  total  volume  in  each  up  to  3  c.c.  are 
now  added. 

Controls. — A  known  positive  and  negative  serum  should  be  included, 
unless  one  is  performing  a  large  number  of  tests  with  reliable  antigens 
every  week,  in  which  case,  among  many  serums,  a  few  at  least  are 
likely  to  be  positive.  Under  these  circumstances  these  controls  may 
be  omitted;  as  a  general  rule,  however,  they  should  be  included. 

To  the  hemolytic  system  control  tube  1  c.c.  of  complement  dilu- 
tion and  2  c.c.  of  salt  solution  are  now  added.  Three  antigen  control 
tubes  are  set  up  for  each  antigen  with  the  dose  employed,  plus  1  c.c. 
of  complement  dilution  and  sufficient  salt  solution  to  make  the  total 
volume  about  3  c.c.  The  corpuscle  control  receives  1  c.c.  of  the  sus- 
pension plus  3  c.c.  of  salt  solution. 

All  the  tubes  are  shaken  gently  and  placed  in  the  incubator  for  an 
hour  at  37°  C.  Instead  of  the  incubator  a  water-bath  at  38°  C.  may  be 
employed  for  one  hour  (not  less)  for  the  primary  and  secondary  periods 
of  incubation,  or  the  primary  period  may  be  conducted  in  a  refrig- 
erator at  8°  C.  for  four  hours  or  over  night.  As  shown  by  McNeal,  the 
latter  method  yields  particularly  delicate  reactions.  In  the  latter 
method  the  secondary  period  of  incubation  is  conducted  in  a  water 
bath  at  38°  C.  for  about  an  hour.  At  the  end  of  primary  incubation 
the  amboceptor  and  corpuscles  are  added  to  each  tube  except  that 
containing  the  corpuscle  control.  Each  tube  is  shaken  gently  and  re- 
incubated  for  an  hour  or  longer,  depending  upon  the  hemolysis  of 
the  controls,  when  the  readings  are  made.  By  making  the  read- 
ings at  this  time  the  influence  of  an  excess  of  hemolysin  due  to 
the  presence  of  natural  antisheep  hemolysin  in  the  human  sera  is. 
avoided  and  lesser  degrees  of  complement  fixation  detected,  which  may 
become  completely  hemolyzed  if  the  tubes  are  set  aside  over  night. 

Reading  the  Results. — The  readings  are  made  in  the  same  manner 
as  described  in  the  first  method,  the  controls  always  being  inspected 
first.  The  hemolytic,  antigen,  and  serum  controls  and  known  nega- 
tive serum  tubes  should  all  be  hemolyzed.  The  antigen  tubes  con- 
taining the  positive  syphilitic  serum  should  not  be  hemolyzed.  Re- 


METHODS    FOR    CONDUCTING   THE    SYPHILIS    REACTION  467 

suits  with  the  unknown  serums  are  dependent  upon  whether  or  not 
the  serums  are  luetic,  and  if  they  are,  upon  the  quantity  of  syphilis 
antibody  present. 

With  strongly  positive  serums  there  is  complete  inhibition  of 
hemolysis  with  all  three  antigens.  With  serums  of  long-standing  or 
treated  cases  of  syphilis  containing  smaller  amounts  of  antibody  the 
reaction  with  the  cholesterinized  extract  is  usually  strongly  positive, 
whereas  with  the  other  two  antigens  the  degree  of  inhibition  of  hemol- 
ysis is  less  marked  and  variable  (see  Fig.  115).  In  from  15  to  20 
per  cent,  of  cases  the  cholesterinized  extract  shows  a  50  per  cent,  or 
more  inhibition  of  hemolysis,  whereas  with  the  other  two  antigens  the 
reactions  are  negative.  In  our  experience  the  majority  of  such 
serums  were  taken  from  patients  giving  a  frank  history  of  syphilis  of 
many  years'  standing  and  from  known  cases  undergoing  treatment, 
further  therapy  being  indicated  until  the  reaction  finally  becomes 
negative  when  cholesternized  extracts  are  used.  In  a  small  pro- 
portion of  cases  a  feebly  positive  reaction  of  25  per  cent,  or  less  in- 
hibition of  hemolysis  may  be  found  with  the  cholesterinized  extract 
alone.  Many  of  these  reactions  occur  with  serums  of  treated  cases 
of  syphilis;  on  the  other  hand,  a  similar  reaction  may  occur  with 
about  5  per  cent,  of  normal  serums,  so  that  if  the  history  and  clinical 
conditions  are  clearly  negative,  a  slight  degree  of  inhibition  of  hemoly- 
sis (5  to  10  per  cent.)  with  the  cholesterinized  extract  and  marked 
hemolysis  with  the  other  two  antigens  may  be  interpreted  as  a  negative 
reaction. 

After  a  new  antigen  has  been  prepared  and  titrated,  it  should  be 
tested  out  in  this  manner  by  placing  it  in  the  series  along  with  at 
least  two  other  older  antigens  of  proved  value,  and  used  in  the  ex- 
amination of  a  large  number  of  serums  before  it  is  finally  accepted  as 
reliable. 

TECHNIC  OF  THE  THIRD  METHOD 
WASSERMANN  REACTION  WITH  VARYING  AMOUNTS  OF  PATIENT'S  SERUM 

As  previously  stated,  it  may  be  desirable  to  measure  the  quantity 
of  syphilis  antibody  in  a  patient's  serum  more  accurately,  especially 
when  observing  the  effect  of  treatment.  This  is  readily  accomplished 
by  using  decreasing  doses  of  serum  with  constant  doses  of  antigen  and 
complement.  In  those  tubes  showing  partial  inhibition  of  hemolysis 
the  degree  of  hemolysis  is  determined  by  comparison  with  a  hemo- 
globin scale  (Boas). 

The  technic  is  quite  similar  to  that  followed  in  the  second  method. 


468 


THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 


1.  One  antigen  is  employed,  either  an  alcoholic  extract  of  syphilitic 
liver  or  acetone-insoluble  lipoids.     The  same  general  rule  as  to  dosage 
is  employed,  namehr,  from  two  to  four  times  the  titrated  antigenic 
unit,    providing   these   amounts   are   not  more    than   one-fourth   the 
anticomplementary  dose. 

2.  0.6  c.c.  of  each  serum  is  heated  to  55°  C.  for  half  an  hour  and 
diluted  1  :  10  by  adding  5.4  c.c.  of  salt  solution. 

3.  Fresh  guinea-pig  serum  complement  is  diluted  1  : 20  as  usual, 
and  used  in  doses  of  1  c.c.  ;  sheep  corpuscles  are  made  up  in  a  2.5  per 
cent,  suspension  and  used  in  doses  of  1  c.c.;   antisheep  amboceptor  is 
adjusted  to  the  complement  and  corpuscles  by  a  method  of  titration, 
as  given  with  the  first  method,  and  used  in  a  dose  equal  to  2  hemo- 
lytic  units. 

The  Test. — Eight  test-tubes  are  arranged  in  a  row  and  the  first 
marked  with  the  patient's  name;  in  each  of  the  first  seven  tubes  the  dose 
of  antigen  is  placed.  The  following  amounts  of  diluted  serum  (1  : 10) 
are  then  added: 


First  tube: 
Second  tube: 
Third  tube: 
Fourth  tube: 
Fifth  tube: 
Sixth  tube: 
Seventh  tube 


0.1  c.c.  serum  diluted  1 
0.2  c.c.  serum  diluted  1 
0.4  c.c.  serum  diluted  1 
0.6  c.c.  serum  diluted  1 
0.8  c.c.  serum  diluted  1 
1.0  c.c.  serum  diluted  1 
2.0  c.c.  serum  diluted  1 
2.0  c.c.  serum  diluted  1 


10  =  0.01  c.c.  serum. 
10  =  0.02  c.c.  serum. 
10  =  0.04  c.c.  serum. 
10  =  0.06  c.c.  serum. 
10  =  0.08  c.c.  serum. 
10  =  0.1  c.c.  serum. 
10  =  0.2  c.c.  serum. 
10  =  0.2  c.c.  serum  (control). 


Eighth  tube: 

The  eighth  tube  contains  no  antigen,  and  is  the  serum  control  with 
the  maximum  dose  employed. 

In  testing  cerebrospinal  fluid  the  following  doses  may  be  used  (undi- 
luted): 0.1  c.c.,  0.2  c.c.,  0.4  c.c.,  0.6  c.c.,  0.8  c.c.,  1  c.c.,  1.5  c.c.,  and 
1.5  c.c.  for  the  eighth  or  control  tube. 

To  each  tu^>e  1  c.c.  of  the  diluted  complement  (1  :  20)  is  now  added, 
and  sufficient  salt  solution  to  bring  the  total  volume  in  each  up  to  3  c.c. 

Controls. — Unless  one  is  examining  a  large  number  of  serums  and 
is  sure  that  the  antigen  is  satisfactory,  known  positive  and  negative 
serum  controls  may  be  included  in  the  series.  As  a  general  rule, 
however,  they  should  not  be  omitted. 

The  hemolytic  system  control  receives  at  this  time  1  c.c.  of  the 
complement  dilution  and  2  c.c.  of  salt  solution.  The  antigen  control 
receives  the  dose  employed  plus  1  c.c.  of  complement  and  sufficient 
salt  solution  to  bring  the  total  volume  up  to  3  c.c.  The  corpuscle 


FIG.  115. — WASSERMANN  REACTION  (SECOND  METHOD). 
Shows  a  +  +  -f  +  reaction  with  the  cholesterinized  extract  (C.  H) ;  a  +  reac- 
tion with  the  alcoholic  extract  of  syphilitic  liver  (S),  and  a  -f-  +  reaction  with  the  ex- 
tract of  acetone-insoluble  lipoids  (A) . 


FIG.  116. — WASSERMANN  REACTION  (THIRD  METHOD). 


METHODS    FOR    CONDUCTING    THE    SYPHILIS    REACTION  469 

control  receives  1  c.c.  of  the  suspension  and  3  c.c.  of  salt  solution,  and 
the  tube  is  plugged  with  cotton  to  show  that  it  is  finished. 

All  tubes  are  shaken  gently  and  incubated  for  one  hour  at  37°  C., 
at  the  end  of  which  time  2  units  of  amboceptor  and  1  c.c.  of  the  cor- 
puscle suspension  are  added  to  all  except  the  corpuscle  control.  Each 
tube  is  shaken  and  all  are  reincubated  for  an  hour  or  an  hour  and  a  half, 
the  length  of  time  depending  upon  the  hemolysis  of  the  serum  controls 
when  the  readings  are  made. 

Reading  the  Results. — As  is  usual,  the  controls  are  examined  first. 
The  hemolytic,  antigen,  negative  serum,  and  all  serum  controls  should 
be  completely  hemolyzed.  The  corpuscle  control  should  show  no 
hemolysis,  indicating  that  the  salt  solution  was  isotonic  and  the  cor- 
puscles free  from  undue  fragility. 

A  1  per  cent,  hemoglobin  solution  in  distilled  water  is  prepared  by 
mixing  1  c.c.  of  the  washed  corpuscles  used  in  preparing  the  suspension 
with  99  c.c.  of  distilled  water.  Into  a  series  of  tubes  dilutions  of 
this  solution  are  placed  as  follows : 

Tube    1 :  10.0  c.c.  of  hemoglobin  solution =  100 

Tube    2:  9.0  c.c.  "            "                "       1.0  c.c.  distilled  water  =90 

Tube    3:  8.0  c.c.  "            "                "      2.0  c.c.  "           "  =80 

Tube    4:  7.0  c.c.  "           "                "      3.0  c.c.  "           "  =70 

Tube    5:  6.0  c.c.  "            "                "      4.0  c.c.  "           "  =60 

Tube    6:  5.0  c.c.  "           "                "      5.0  c.c.  "           "  =50 

Tube    7:  4.0  c.c.  "       .     "                "      6.0  c.c.  "           "  =40 

Tube    8:  3.0  c.c.  "           "                "      7.0  c.c.  "           "  =  30 

Tube    9:  2.0  c.c.  "           "                "      8.0  c.c.  "           "  =20 

Tube  10:  1.0  c.c.  "           "                "      9.0  c.c.  "           "  =  10 

Tube  11:  0.4  c.c.  "            "                "      9.6  c.c.  «'•'        "  =4 

Tube  12: 10.0  c.c.  =    0 

This  scale  is  not  permanent,  and  must  be  prepared  anew  for  each 
set  of  reactions. 

A  negative  reaction  is  indicated  in  the  seventh  tube,  which  contains 
the  maximum  dose  of  serum  (0.2  c.c.),  by  complete  hemolysis  or  by 
hemolysis  ranging  from  80  to  100,  according  to  the  scale.  Inhibition 
of  hemolysis  in  this  tube  giving  a  hemoglobin  scale  ranging  from  70 
to  4  (inclusive)  is  regarded  as  a  positive  reaction.  Absolute  lack  of 
hemolysis  in  this  tube  is  0  according  to  the  scale,  but  usually  the 
patient's  serum  or  complement  serum  is  sufficiently  tinged  with  hemo- 
globin to  give  a  slight  color  to  the  supernatant  fluid,  so  that  an  Ab- 
solute positive  reaction  may  range  from  0  to  4.  For  instance,  the 
serum  of  an  untreated  case  of  secondary  syphilis  gave  the  following 
reading  (see  Fig.  117). 


470  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 


Tube  1 
Tube  2 
Tube  3 
Tube  4 
Tube  5 
Tube  6 
Tube  7 
Tube  8 


0.01  c.c.  serum 100 

0.02  c.c.  serum 100 

0.04  c.c.  serum 30 

0.06  c.c.  serum 10 

0.08  c.c.  serum 4 

0.1     c.c.  serum 4 

0.2     c.c.  serum 2 

0.2     c.c.  serum  (control) 100 


.  Although  these  figures  suffice  for  recording  purposes,  they  are  not 
adapted,  without  some  qualifying  phrase,  for  rendering  reports  to 
clinicians.  According  to  the  inhibition  of  hemolysis  or  the  degree  of 
hemolysis  in  the  sixth  tube  containing  the  maximum  quantity  of 
serum  the  following  scheme  may  be  used  in  reporting  the  results  : 


0  to  10  =  strongly  positive  = 
10  to  30  =  moderately  positive  = 
30  to  50  =  slightly  positive  =  (  +  +) 
50  to  80  =  very  weakly  positive  =  (+) 
80  to  90  =  doubtful  or  delayed  hemolysis  =(=*=) 
100  =  negative  =  (  —  ) 

TECHNIC  OF  THE  FOURTH  METHOD 
WASSERMANN  REACTION  WITH  VARYING  AMOUNTS  OF  COMPLEMENT 

In  this  method  the  amount  of  syphilis  antibody  in  a  serum  is  meas- 
ured according  to  the  number  of  hemolytic  doses  of  complement  ab- 
sorbed or  fixed  with  a  constant  amount  of  antigen.  As  previously 
stated,  any  organic  extract  used  as  antigen  may  of  itself  fix  a  certain 
amount  of  complement;  a  non-syphilitic  serum  may  do  the  same, 
and  a  mixture  of  the  two  may  fix  still  more,  though  the  amounts  may 
be  relatively  small.  A  peculiarity  possessed  by  a  syphilitic  serum  is 
that  it  fixes  a  large  amount  of  complement  when  mixed  with  antigen; 
as  a  result  the  test  becomes  a  quantitative  and  not  a  qualitative  reac- 
tion. The  only  practical  means  we  possess  of  estimating  the  amount 
of  complement  in  a  fresh  serum  is  to  ascertain  the  hemolytic  dose, 
i.  e.,  to  find  the  smallest  quantity  of  serum  that  is  just  sufficient  com- 
pletely to  lyse  the  test  amount  of  corpuscles  with  the  hemolysin.  When 
this  has  been  done,  the  quantity  of  complement  used  in.  the  reaction 
may  be  expressed  in  terms  of  hemolytic  doses  fixed,  and  not  in  terms 
of  the  amount  of  fresh  serum. 

When  properly  performed  according  to  this  method,  which  has 
been  modified  after  the  technic  of  Browning  and  Mackenzie1  and 
1  Ztschr  f.  Immunitatsf.,  orig.,  1909,  2,  459. 


METHODS    FOR    CONDUCTING   THE    SYPHILIS    REACTION  471 

Thomsen,1  the  syphilis  reaction  becomes  quite  delicate  and  accurate, 
but  is  more  complicated  than  the  other  methods,  and  should  not  be 
attempted  until  one  is  accustomed  to  the  simpler  test  and  thoroughly 
understands  the  underlying  principles  of  the  syphilis  reaction  and  knows 
the  many  sources  of  fallacy.  The  greater  amount  of  work  that  it  en- 
tails and  the  larger  quantities  of  complement-serum  and  amboceptor 
that  are  required  may  serve  as  factors  against  its  adoption  as  a  routine 
method.  On  the  other  hand,  the  sources  of  error  are  well  under  control, 
and  the  test  has  yielded  remarkably  uniform  results  in  the  hands  of  my 
colleagues  in  their  respective  laboratories,  working  with  the  serums 
of  the  same  persons. 

The  technic  as  given  here  has  been  worked  out  and  tested  with  a 
large  number  of  sera  by  Matsunami,  Brown,  Meine  and  I  in  an  effort 
to  evolve  a  standardized  Wassermann  reaction.  Every  phase  of  the 
Wassermann  reaction  has  been  studied  specifically  by  experiment  and 
the  following  technic  is  based  upon  these  results: 

1.  Corpuscles. — Sheep  corpuscles  are  washed  three  times  with  salt 
solution  and  made  up  in  a  2|  per  cent,  suspension;  dose,  0.5  c.c: 

2.  Hemolysin. — Antisheep  hemolysin  is  titrated  by  placing  increas- 
ing amounts  of  diluted  serum,  as  0.1  c.c.,  0.15  c.c.,  0.25  c.c.,  0.30  c.c., 
and  0.35  c.c.  of  a  1  : 300  dilution  in  a  series  of  test-tubes  and  adding 
to  each  tube  0.025  complement  serum  (=  0.5  c.c.  of  a  1  :  20  dilution), 
0.5  c.c.  of  2J  per  cent,  corpuscle  suspension,  and  sufficient  salt  solution 
to  make  the  total  volume  in  each  tube  about  3  c.c.    Each  tube  is  gently 
shaken  and  incubated  in  the  water-bath  at  38°  C.  for  one  hour,  when  the 
reading  is  made.    The  unit  of  hemolysin  is  the  smallest  amount  giving 
complete  hemolysis.     Instead  of  using  increasing  amounts  of  one  di- 
lution of  hemolysin  as  above,  a  series  of  dilutions  may  be  made  in  flasks, 
as  1  :  2000;  1  :  2500;  1  : 3000;  1  : 3500;  1  : 4000,  etc.,  and  1  c.c.  of  each 
used  in  the  titration. 

The  unit  need  not  be  determined  more  than  once  in  two  weeks, 
providing  the  hemolysin  is  kept  at  a  low  temperature. 

3.  Complement. — It  is  advisable  to  use  the  mixed  sera  of  at  least 
two  or  more  healthy  guinea-pigs.    When  only  a  small  amount  of  com- 
plement serum  is  required,  sufficient  blood  may  be  obtained  by  aspirat- 
ing 2  c.c.  of  blood  from  the  hearts  of  several  large  animals  (see  page  41). 
The  complement  serum  should  be  clear,  collected  a  few  hours  before  use, 
and   preferably  from  fasting   animals.     The   mixed   serum  is  diluted 
with  9  parts  of  normal  salt  solution  (1  :  10)  and  titrated.     This  titra- 

1  Ztschr.  f.  Immunitatsf.,  orig.,  1910,  7,  389. 


472 


THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 


tion  must  be  performed  with  every  complement  serum  before  the  main 
tests  are  conducted.  In  order  to  allow  for  the  anticomplementary 
action  of  the  antigen  the  complement  is  titrated  as  suggested  by  Thorn- 
sen,  in  the  presence  of  the  dose  of  antigen,  as  determined  by  titration, 
to  be  used  in  the  main  tests,  as  follows:  In  each  of  a  series  of  six  test- 
tubes  place  the  dose  of  antigen  (as,  for  example,  0.1  c.c.  of  1  :  20  dilution 
of  acetone  insoluble  lipoids) ;  add  the  following  amounts  of  complement 
1  :  10  with  an  accurate  pipet:  0.1  c.c.,  0.2  c.c.,  0.3  c.c.,  0.4  c.c.,  0.5  c.c., 
and  0.6  c.c.  Add  sufficient  salt  solution  to  make  the  total  volume  in 
each  tube  about  2  c.c.,  mix  gently  and  incubate  in  a  water-bath  at  38°  C. 
for  one  hour,  then  one  unit  of  hemolysin  and  0.5  c.c.  of  2|  per  cent, 
suspension  of  cells  are  added,  the  tubes  shaken  and  reincubated  in  the 
water-bath  for  an  hour,  when  the  reading  is  made.  The  unit  of  comple- 
ment is  the  smallest  amount  producing  complete  hemolysis.  The  results 
of  a  titration  are  shown  in  Table  15a  and  Fig.  110. 

TABLE  15a.— RESULTS  OF  COMPLEMENT  TITRATION  IN  THE  PRES- 
ENCE OF  ANTIGEN 


TUBE 

ANTIGEN 
1  :20, 

COMPLE- 
MENT 

«;-S  § 

HEMOL- 

CELLS 2£ 
PER  CENT., 

.S| 

RESULTS 

No. 

C.c. 

1  :  10, 
C.c. 

<N-£  ° 

YSIN 

C.c. 

J° 

^H   0 
11" 

a|o 

hj 

o 

Id 

1 

0.2 

0.1 

1  unit 

0.5 

Very  weak  hemol- 

ysis 

2 

0.2 

0.2 

"^-doo 

1  unit 

0.5 

fl  °^ 

Marked  hemolysis 

3 

0.2 

0.3 

o  c^00 

1  unit 

0.5 

cj  CO 

Almost     complete 

'is       ta 

-^ts 

hemolysis 

4 

0.2 

0.4 

"o-^ 

1  unit 

0.5 

^ 

Complete       hemol- 

^ d  ^ 

S)"5 

ysis;  the  unit 

5 

0.2 

0.5 

1  "^ 

1  unit 

0.5 

«  ja 

Complete     hemol- 

"O   O>    i      . 

^  i   . 

ysis 

6 

0.2 

0.6 

S^"!  ^ 

1  unit 

0.5 

^•g  § 

Complete     hemol- 

*_f 

10  sj 

ysis 

4.  Antigen. — A  large  number  of  comparative  tests  with  the  same 
sera  and  a  variety  of  antigens  have  shown  that  a  good  extract  of  acetone- 
insoluble  lipoids  is  best  adapted  for  this  reaction.  As  a  general  rule  these 
extracts  possess  a  marked  degree  of  antigenic  sensitiveness  and  are  us- 
ually free  of  anticomplementary  action  except  in  very  large  doses.  Each 
extract  must  be  titrated  to  determine  the  proper  dose  to  employ;  as  a 
general  rule  it  is  sufficient  to  titrate  an  extract  once  every  two  months 
providing  it  is  carefully  refrigerated  and  is  yielding  satisfactory  results 
in  complement-fixation  tests. 

In  conducting  these  titrations  a  mixed  complement  serum  diluted 
1  : 10  is  titrated  in  the  following  doses  with  one  unit  of  hemolysin  and 


METHODS   FOR   CONDUCTING   THE    SYPHILIS   REACTION  473 

0.5  c.c.  of  a  2J  per  cent,  suspension  of  cells:  0.1  c.c.,  0.15  c.c.,  0.2  c.c., 
0.25  c.c.,  0.3  c.c.,  and  0.4  c.c.  In  the  antigenic  titration  the  following 
amounts  of  antigen  diluted  1  :  20  are  placed  in  a  series  of  ten  test-tubes: 
0.001  c.c.,  0.005  c.c.,  0.008  c.c.,  0.01  c.c.,  0.05  c.c.,  0.08  c.c.,  0.1  c.c., 
0.2  c.c.,  0.3  c.c.,  and  0.4  c.c.;  0.1  c.c.  of  fresh  inactivated  syphilitic  serum 
preferably  from  several  persons  mixed,  plus  2  units  of  complement 
and  sufficient  salt  solution  to  make  3  c.c.,  are  added  to  each  tube  and 
incubated  in  a  water-bath  at  38°  C.  for  one  hour,  when  1  unit  of  hemol- 
ysin  and  0.5  c.c.  of  corpuscles  are  added,  tubes  shaken,  and  re-incubated 
for  an  hour.  The  reading  may  be  made  at  once  or  after  the  corpuscles 
have  settled;  the  unit  is  the  smallest  amount  of  antigen  yielding  complete 
inhibition  of  hemolysis. 

In  the  anticomplementary  titration  the  extract  is  diluted  1  : 20  and 
the  following  amounts  placed  in  a  series  of  ten  test-tubes  with  0.1  c.c. 
of  fresh  inactivated  normal  serum  and  2  units  of  complement:  0.1, 
0.2,  0.3,  0.5,  0.8,  1.0,  1.2,  1.5,  and  2.0  c.c.  The  titration  is  conducted 
in  the  same  manner,  and  the  anticomplementary  unit  is  the  smallest  amount 
of  extract  producing  inhibition  of  hemolysis. 

Hemolytic  and  serum  controls  on  both  the  syphilitic  and  normal 
serum  should  be  included  and  show  complete  hemolysis. 

A  satisfactory  extract  is  one  in  which  the  antigenic  unit  is  at  least 
ten  times  less  than  the  anticomplementary  unit,  and  in  conducting  the 
Wassermann  reaction  2  units  of  antigen  are  employed  as  the  dose. 

5.  Fluid  to  be  Tested.— Serum  is  heated  at  56°  C.  for  half  an  hour 
and  used  in  dose  of  0.1  c.c.    Cerebrospinal  fluid  should  be  fresh  and  is 
used  unheated  in  dose  of  1  c.c. 

6.  The  Test. — For  each  serum  eight  test-tubes  are  arranged  in  a  row. 
One  is  labeled  with  the  patient's  name  and  all  are  numbered.     Into 
each  is  placed  0.1  c.c.  of  the  patient's  serum.     Into  each  of  the  first 
six  tubes  are  placed  the  dose  of  antigen  and  2,  3,  4,  5,  6,  and  8  units  of 
complement  respectively.    The  last  two  tubes  are  the  serum  controls, 
to  determine  the  amount  of  complement  fixed  by  serum  alone,  and  re- 
ceive 1  and  2  units  of  complement  respectively.    Sufficient  salt  solution 
is  added  to  each  tube  to  bring  the  total  volume  up  to  3  c.c.,  and  all  are 
incubated  in  the  water-bath  at  38°  C.  for  an  hour.    One  unit  of  hemolysin 
and  0.5  c.c.  of  corpuscle  suspension  are  now  added  to  each  tube  and  re- 
incubated  in  the  water-bath  for  an  hour,  when  the  readings  are  made. 
Sharper  readings  may  be  made  after  the  corpuscles  have  settled  either  by 
centrifuging  or  standing  the  tubes  in  the  refrigerator  overnight. 

A  human  hemolytic  system  may  be  employed  if  a  sufficiently  potent 


474 


THE    TECHNIC    OF    COMPLEMENT-FIXATION   REACTIONS 


antihuman  hemolysin  is  at  hand.  Comparative  tests  using  the  anti- 
sheep  and  antihuman  hemolytic  systems  with  the  same  sera  have  shown 
that  the  degree  of  inhibition  of  hemolysis  with  positive  sera  is  occasion- 
ally greater  with  the  antihuman  system. 

Controls. — The  anticomplementary  action  of  each  serum  is  controlled 
in  the  last  two  tubes  of  each  series;  unless  a  serum  is  markedly  anti- 
complementary,  the  use  of  1  and  2  units  of  complement  is  sufficient. 

2.  A  known  positive  and  negative  serum  may  be  included. 

3.  A  hemolytic  control  is  set  up  with  1  unit  of  complement  and 
antigen;  after  the  primary  incubation  1  unit  of  hemolysin  and  the  cells 
are  added.    This  tube  is  a  control  on  the  unit  of  complement  and  should 
show  complete  hemolysis. 

4.  A  corpuscle  control  may  be  included,  containing  0.5  c.c.  of  cor- 
puscles and  3  c.c.  of  salt  solution.    It  controls  the  tonicity  of  the  salt 
solution  and  should  show  no  hemolysis. 

Reading  the  Results. — The  controls  are  first  inspected.  The  corpus- 
cle control  should  show  no  hemolysis  and  the  hemotytic  control  be  just 
hemolyzed.  The  last  two  tubes  of  each  series  are  the  serum  control  tubes, 
and  the  first  tube  containing  1  unit  of  complement  may  show  incomplete 
hemolysis,  while  the  second  tube  containing  2  units  of  complement 
shows  complete  hemolysis  unless  the  serum  is  quite  anticomplementary. 

TABLE   15b.— RESULTS   AND    METHOD   OF   READING   THE   WASSER- 
MANN  REACTION    (QUANTITATIVE   METHOD) 


SERUM 

RESULTS  OF  COMPLEMENT  FIXATION  TESTS 

CONTROLS 

AMOUNT  OF 

DIAG- 

No. 

NOSIS 

2  Units 

3  Units 

4  Units 

5  Units 

6  Units 

8  Units 

1  Unit 

2Units 

ABSORBED; 
READINGS 

1 

Normal 















_ 

Negative 

2 
3 

Normal 
Syphilis 

+  +  +  + 

+  +  +  + 

+  +  + 

~ 

I 

— 

db' 

— 

Negative 
Positive;  4 

units 

4 

Syphilis 

+  +  + 

-)--)- 

db 

— 

— 

— 

=fc 

— 

Positive;  3 

units 

5 

Syphilis 

_|_-|_ 

_ 

_ 

_ 

_ 

_ 

— 

— 

Positive;  2 

units 

6 

Svohilis 

+  +  +  + 

+  +  +  + 

+  +  +  + 

_!__!_  _|__|_ 

+  +  +  + 

+  +  +  + 

_|. 

_ 

Positive;  at 

least  8  units 

7     Syphilis 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+  +  +  + 

+ 

* 

Positive;  at 
least  8  units 

8      Syphilis 

+  +  +  + 

_}--j- 

4. 

— 

— 

— 

+ 

± 

Positive;  2  to 

3  units 

The  first  six  tubes  of  each  series  show  whether  or  not  the  reaction  is 
positive  or  negative,  and  if  positive,  the  amount  of  complement  absorbed. 
If  tube  No.  7  of  the  serum  controls  shows  some  inhibition  of  hemolysis, 
1  unit  is  subtracted  from  the  number  of  units  of  complement  absorbed 
in  the  first  six  tubes,  and  the  difference  represents  the  amount  of  comple- 
ment absorbed  by  antigen  and  syphilitic  antibody.  /  regard  the  reaction 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  475 

as  positive  when  lysis  is  incomplete  vrith  2  units  of  complement,  in  addition 
to  the  amount  absorbed  by  the  serum  alone.  More  strongly  reacting  serums 
will  absorb  from  3  to  8  units  of  complement,  and  not  infrequently  more; 
these  sera  may  be  retested  with  8,  10,  12,  etc.,  units  of  complement, 
but  the  employment  of  these  large  doses  routinely  is  not  justified  because 
of  the  large  amount  of  complement  used,  and  the  complement-fixing 
power  of  the  majority  of  sera  are  readily  measured  with  2  to  8  units. 
The  method  of  reading  is  best  shown  in  Table  15b  and  Fig.  117. 

Cases  of  syphilis  progressing  favorably  with  the  administration  of 
specific  remedies  show  less  and  less  complement  fixation  until  a  negative 
reaction  is  secured.  A  large  number  of  comparative  tests  employing 
the  original  Wassermann  and  this  reaction  with  the  same  antigen  have 
shown  that  a  serum  yielding  a  -f  +  +  +  result  in  the  original  Wasser- 
mann reaction  may  show  anywhere  from  2  to  8  units  of  fixation  with  this 
technic. 


MODIFICATIONS  OF  THE  WASSERMANN  REACTION 
MODIFICATION  OF  NOGUCHI 

Among  the  large  number  of  modifications  of  the  original  syphilis 
reaction  that  have  been  devised,  that  of  Noguchi  has  proved  of  dis- 
tinct value.  In  this  method  an  antihuman  hemolytic  system  is 
employed  that  eliminates  one  possible  source  of  error,  due  to  the 
natural  antisheep  amboceptor  in  human  serum. 

Noguchi  advocated  the  use  of  active  serum  for  this  test,  with  an 
antigen  of  acetone-insoluble  lipoids.  Active  serum  yields  a  more 
delicate  reaction,  but  may  give  false  or  proteotropic  complement 
fixation,  especially  when  crude  alcoholic  extracts  are  used  as  antigens. 
Before  I  began  using  cholesterinized  alcoholic  extracts  in  making  the 
Wassermann  reaction  I  not  infrequently  found  that  the  Noguchi  test 
with  active  serum  was  more  delicate  than  the  Wassermann  reaction, 
but  with  cholesterinized  extracts  the  results  ran  closely  parallel.  It 
is  a  good  practice  to  conduct  both  a  Wassermann  and  a  Noguchi  test 
with  each  serum,  as  a  negative  Noguchi  test  with  active  serum  is  a 
better  indication  of  the  absence  of  syphilis  than  is  a  negative  Wasser- 
mann reaction.  A  positive  Wassermann  reaction,  however,  is  better 
evidence  of  the  presence  of  syphilis  than  is  a  positive  Noguchi  reaction, 
because  of  the  possibility  of  false  complement  fixation  occurring  in 
the  latter  when  active  serums  are  used.  I  may  state,  however,  that 
when  a  good  antigen  of  acetone-insoluble  lipoids  is  used,  the  per- 
centage of  false  reactions  is  relatively  small,  being  less  than  2  per 


476  THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

cent.  The  Noguchi  test,  on  the  other  hand,  may  be  conducted  with 
inactivated  serums,  when  the  danger  of  false  reactions  is  removed, 
but  the  delicacy  of  the  test  is  likewise  diminished,  so  that  it  more 
closely  approaches  the  Wassermann  reaction. 

Noguchi  endeavored  to  simplify  the  technic  of  the  syphilis  reaction 
by  preparing  complement,  antigen,  and  amboceptor  dried  on  filter- 
paper.  These  were  titrated  and  so  adjusted  that  a  certain  measure 
of  paper  represented  the  required  amount  of  each  reagent.  In  this 
manner  it  would  be  possible  for  a  large  central  laboratory  to  prepare 
and  standardize  these  reagents,  putting  them  up  ready  for  use  in  the 
simplest  possible  form,  and  thus  making  them  available  for  the 
practising  physician.  Complement,  however,  deteriorates  so  rapidly 
that  the  paper  is  useless  unless  it  is  freshly  prepared.  The  antigen 
slips  likewise  deteriorate,  but  not  so  rapidly  as  the  complement;  the 
amboceptor,  however,  is  well  preserved  by  this  method,  and  forms 
a  simple  method  for  titrating  and  handling  this  important  reagent. 

Technic  of  the  Noguchi  Modification. — (1)  Complement  is  fur- 
nished by  the  fresh,  clear  serum  of  the  guinea-pig,  put  up  in  40  per 
cent,  solution,  prepared  by  diluting  1  part  of  serum  with  1J^  parts  of 
normal  salt  solution.  Dose  0.1  c.c.  (5  drops  from  a  capillary  pipet). 
Whenever  possible  it  is  always  best  to  use  a  mixture  of  the  serums 
from  two  or  more  guinea-pigs. 

2.  Human  Corpuscles. — These  are  washed  three  times  with  normal 
salt  solution,  and  used  in  dose  of  1  c.c.  of  a  1  per  cent,  suspension. 
To  a  graduated  centrifuge  tube  containing  9  c.c.  of  sterile  2  per  cent, 
sodium  citrate  in  normal  salt  solution  add  1  c.c.  of  blood  and  shake 
gently.     This  amount  of  blood  is  easily  secured  by  pricking  the  finger 
with  a  lancet  and  collecting  the  blood  in  the  centrifuge  tube  up  to  the 
mark  10.     This  is  centrifuged  thoroughly,  and  the  supernatant  fluid 
drawn  off.     More  saline  solution  is  then  added  to  the  corpuscles  stirred 
up  and  the  mixture  centrifuged.     The  washing  is  repeated  once  more, 
and  the  supernatant  fluid  discarded.     The  corpuscles  are  then  sus- 
pended in  sufficient  salt  solution  to  make  a  total  volume  of  100  c.c. 

3.  Hemolytic  Amboceptor. — Antihuman  hemolysin   is   prepared  by 
immunizing  rabbits  with  human  cells,  as  described  on  p.  72.     It  is  a 
difficult  matter  to  secure  a  potent  amboceptor;    human  erythrocytes 
are  more  toxic  than  sheep's  cells  for  rabbits,  and  most  animals  produce 
but  small  amounts  of  the  amboceptor.     Hemagglutinins  are  produced  inV 
large  amounts,  and  when  using  a  low  titer  hemolytic  serum,  the  test- 
corpuscles  are  quickly  agglutinated  in  small  dense  masses  that  are 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  477 

broken  up  with  difficulty  and  that  interfere  greatly  with  hemolysis. 
With  serums  having  a  titer  of  1  :  1000  or  over,  the  agglutinins  are  not 
so  much  in  evidence;  a  satisfactory  reaction  is  best  observed,  therefore, 
with  a  potent  amboceptor  (1  :  1000)  serum. 

The  hemolytic  serum  may  be  preserved  in  1  c.c.  ampules  after 
adding  an  equal  part  of  glycerin,  and  a  stock  dilution  prepared  and 
titrated  in  the  usual  manner.  The  serum  is  also  well  preserved  dried 
on  filter-paper,  as  described  on  p.  80.  A  trial  titration  should  always 
be  made  to  determine  the  potency  of  the  serum  before  the  paper  slips 
are  prepared. 

//  paper  amboceptor  is  used,  the  uniform  rule  of  titrating  it  with  the 
complement  and  corpuscles  on  hand  should  be  observed  before  the  actual 
tests  are  made.  This  is  done  chiefly  because,  where  one  guinea-pig 
serum  is  used  for  complement,  it  may  occasionally  happen  that  the 
serum  is  less  active  than  usual,  so  that  if  fixed  doses  of  complement  and 
amboceptor  are  used,  the  reactions  may  at  times  prove  to  be  incomplete 
and  inaccurate.  The  process  of  titration  is  so  simple  that  any  one  may 
readily  conduct  it,  and  thus  fulfil  the  most  important  requirement  of  any 
complement-fixation  test,  namely,  adjustment  of  the  complement, 
amboceptor,  and  corpuscles  to  one  another. 

Titration  of  Serum  Hemolysin. — Prepare  a  1 : 100  dilution  by  mixing 
0.1  c.c.  of  immune  serum  (inactivated)  with  9.9  c.c.  of  saline  solution. 
To  a  series  of  six  small  test-tubes  add  increasing  amounts  of  this  diluted 
serum  as  follows: 

Tube  1:  0.1  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  2:  0.2  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  3:  0.4  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  4:  0.5  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  5:  0.8  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  6:  1  c.c.  amboceptor  serum  (1  :  100) +0.1  c.c.  complement 

(40  per  cent.)  +  l  c.c.  corpuscle  suspension  (1  per  cent.). 

Sufficient  saline  solution  is  added  to  the  first  tubes  of  the  series  to 
bring  the  total  volume  up  to  2  c.c.  The  tubes  are  then  shaken  gently 
and  placed  in  the  incubator  at  37°  C.  for  two  hours  (or  one  hour  in 
water-bath  at  the  same  temperature) ,  during  which  time  they  should  be 
inspected  and  shaken  gently  several  times.  At  the  end  of  the  period  of 
incubation  that  tube  which  shows  just  complete  hemolysis  contains  one 


478  THE   TECHNIC    OF    COMPLEMEMT-FIXATION    REACTIONS 

amboceptor  unit,  and  double  this  amount  is  used  in  making  the  main 
tests.  If  the  serum  has  a  titer  of  less  than  1  :  500,  it  should  not  be  used 
either  in  preparing  the  amboceptor  slips  or  in  conducting  the  reaction. 
Titration  of  Dried  Amboceptor  Paper. — After  the  paper  (S.  &  S.  No. 
597)  has  been  evenly  saturated  with  immune  serum  and  dried,  the  sheets 
are  cut  into  5  mm.  strips  and  standardized  by  placing  increasing  lengths 
of  paper  into  a  series  of  tubes  as  follows: 

Tube  1:  1  mm, paper +0.1  c.c.  complement  (40  per  cent.)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  2:  2  mm.  paper +0.1  c.c.  complement  (40  per  cent.)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  3:  3  mm.  paper+0.1  c.c.  complement  (40  per  cent.)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  4:  4  mm.  paper+0.1  c.c.  complement  (40  per  cent.)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  5:  5  mm.  paper+0.1  c.c.  complement  (40  per  cent.)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 
Tube  6:  6  mm.  paper+0.1  c.c.  complement  (40  per  cento)  (5  drops) 

+  1  c.c.  corpuscle  suspension  (1  per  cent.). 

One  cubic  centimeter  of  saline  solution  is  added  to  each  tube,  and 
the  mixture  shaken  gently  and  incubated  at  37°  C.  for  two  hours  or 
one  hour  in  a  water-bath.  At  the  end  of  this  time  the  tube  just  completely 
hemolyzed  contains  one  amboceptor  unit,  and  in  performing  the  test  double 
this  amount  is  used.  (See  Fig.  118). 

This  titration  should  always  be  conducted  before  the  actual  tests 
are  set  up,  as  is  the  rule  in  conducting  the  Wassermann  reaction.  When 
the  titer  of  the  paper  is  known,  it  may  not  be  necessary  to  set  up  all  the 
tubes  of  the  foregoing  series,  as  a  few  only  are  necessary  to  determine 
if  the  same  amount  of  paper  as  was  used  in  the  previous  tests  will  suffice 
with  the  new  complement  and  corpuscle  suspension  at  hand. 

All  titrations  and  the  main  tests  may  be  conducted  in  a  water-bath 
(37°  C.).  With  the  aid  of  a  good  thermometer  a  satisfactory  bath  is 
easily  improvised.  In  fact,  I  believe  that  better  results  are  secured 
in  the  water-bath  than  in  the  incubator.  It  is  possible,  therefore,  to 
conduct  these  reactions  in  a  perfectly  satisfactory  manner  without  the 
aid  of  an  expensive  incubator. 

4.  Antigen. — Acetone-insoluble  lipoids  (Noguchi)  are  to  be  used 
exclusively  if  the  tests  are  conducted  with  active  serums.  When  heated 
serums  are  used,  any  extract  may  be  employed,  as  in  making  the  Wasser- 
mann reaction,  but  the  same  antigen  gives  excellent  results,  and  I  use  it 
exclusively  in  conducting  the  Noguchi  reaction  with  both  active  and 
inactivated  serums. 


FlG.    118. TlTRATION    OF    ANTIHUMAN    HEMOLYTIC    AMBOCEPTOR. 

Shows  complete  hemolysis  with  4  mm.  of  paper  ( =  unit). 


IZ 


tt  II 


:; 


II 

I 


FIG.  119. — NOGUCHI  MODIFICATION  OF  THE  WASSERMANN  REACTION. 
Positive  reactions  in  tubes  1  and  3  (see  Table  16). 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  479 

The  antigen  must  be  titrated  as  usual,  and  its  anticomplemen- 
tary  hemolytic  and  antigenic  properties  determined.  According 
to  Noguchi,  an  extract  is  satisfactory  if  it  is  antigenic  in  0.02  c.c. 
of  a  1  :  10  dilution,  and  not  anticomplementary  or  hemolytic  in 
amounts  under  0.4  c.c.  (1  :  10).  In  conducting  the  tests  five  times 
the  antigenic  unit,  or  0.1  c.c.,  is  employed;  this  dose  is  at  least  four 
times  smaller  than  the  anticomplementary  unit,  and  is  therefore  safe 
and  satisfactory. 

The  antigen  is  best  preserved  in  methyl  alcohol,  as  described  on 
p.  446.  Dried  on  paper  and  properly  preserved  in  sealed  tubes  in  a  cold 
place  it  will  retain  its  activity  for  several  months,  but  as  a  general  rule 
it  is  best  to  use  fresh  emulsions  of  the  alcoholic  solution. 

Titration  of  Antigen. — The  anticomplementary,  hemolytic,  and 
antigenic  doses  of  an  extract  are  determined  in  the  same  general  manner 
as  was  described  under  the  Wassermann  reaction. 

1.  Anticomplementary  Titration. — A  portion  of  the  stock  alcoholic 
solution  of  acetone-insoluble  lipoids  is  diluted  with  9  parts  of  saline 
solution.     This  is  the  emulsion  that  is  employed  in  conducting  the 
Noguchi  reaction,  and  contains  0.3  per  cent,  of  the  original  lipoidal 
substances. 

Sufficient  emulsion  for  these  titrations  is  prepared  by  diluting  0.4 
c.c.  of  the  alcoholic  solution  with  3.6  c.c.  of  saline  solution. 

Into  a  series  of  seven  small  test-tubes  place  increasing  amounts  of 
this  emulsion  as  follows:  0.1,  0.2,  0.3,  0.4,  0.6,  0.8,  1.0  c.c.,  add  0.1  c.c. 
(5  capillary  drops)  complement  (40  per  cent.)  to  each;  also  1  c.c.  of  a 
1  per  cent,  suspension  of  corpuscles  and  sufficient  saline  solution  to 
make  the  total  volume  in  each  tube  about  2  c.c.  Incubate  at  37°  C. 
for  one  hour  (one-half  hour  in  water-bath),  and  add  two  units  of  ambo- 
ceptor.  Shake  the  tubes  gently  and  reincubate  for  two  hours  (one  hour 
on  water-bath).  That  tube  showing  beginning  inhibition  of  hemolysis 
contains  the  anticomplementary  dose,  which  should  not  be  under  0.4 
c.c.  In  the  tubes  containing  the  larger  doses  slight  hemolysis  may  be 
noticed,  which  is  evidence  of  the  hemolytic  action  of  the  extract. 

An  eighth  tube  should  be  included,  containing  0.1  c.c.  diluted  com- 
plement, two  units  of  amboceptor,  and  1  c.c.  of  the  corpuscle  suspension. 
This  is  the  hemolytic  control  and  should  show  complete  hemolysis. 

2.  Antigenic  Titration. — Since  the  extract  is  likely  to  have  a  high 
antigenic  value,  it  is  necessary  to  dilute  the  antigen  still  further  by  plac- 
ing 0.5  c.c.  of  the  foregoing  emulsion  in  a  test-tube  and  .adding  4.5  c.c. 
of  saline  solution  (1  :  100  dilution  of  the  antigen).     Into  a  series  of  six 
test-tubes  place  increasing  amounts  of  this  emulsion  as  follows:    0.05, 


480  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

0.1,  0.2,  0.3,  0.4,  0.5  c.c.  To  each  tube  add  four  drops  (0.08  c.c.)  of 
inactivated  or  one  drop  (0.02  c.c.)  of  fresh  active  syphilitic  serum;  also 
0.1  c.c.  (five  capillary  drops)  of  complement  (40  per  cent.)  and  1  c.c.  of 
1  per  cent,  corpuscle  suspension.  Then  add  sufficient  salt  solution  to 
bring  the  total  up  to  2  c.c. 

Two  controls  should  be  included:  (1)  The  serum  control,  containing 
the  dose  of  serum,  0.1  c.c.  of  the  complement,  1  c.c.  of  corpuscle  sus- 
pension, and  saline  solution;  (2)  the  hemolytic  control,  containing  at 
this  time  0.1  c.c.  of  complement,  1  c.c.  of  corpuscle  suspension,  and 
sufficient  saline  solution. 

All  tubes  are  incubated  for  one  hour  at  37°  C.  (one-half  hour  in 
water-bath),  after  which  two  units  of  amboceptor  are  added  to  each 
tube.  The  tubes  are  then  shaken  gently  and  reincubated  for  two  hours 
(one  hour  in  water-bath).  At  the  end  of  this  time  the  two  controls 
should  be  completely  hemolyzed,  and  in  the  series  proper  that  tube 
showing  just  complete  inhibition  of  hemolysis  contains  one  antigenic 
unit.  Usually  the  first  and  second  tubes  show  some  inhibition  of 
hemolysis,  and  in  the  third  and  other  tubes  hemolysis  is  completely 
inhibited.  In  this  case  0.2  c.c.  of  this  emulsion  would  be  one  anti- 
genic unit  ( =  0.02  c.c.  undiluted  antigen)  •  five  times  this  amount  equals 
0.1  c.c.  of  the  first  emulsion  (1:10),  which  is  the  amount  to  be  used  in 
making  the  main  tests. 

Unless  the  antigen  shows  signs  of  deterioration,  these  titrations  need 
be  made  only  about  once  a  month. 

If  paper  antigen  is  employed,  both  titrations  are  conducted  in 
exactly  the  same  manner  by  adding  increasing  lengths  of  a  strip  of 
dried  paper  5  mm.  in  width,  impregnated  with  the  antigen. 

5.  Fluid  to  be  Tested. — If  active  serum  is  used,  it  should  be  fresh, 
free  from  hemoglobin,  and  preferably  not  over  twenty-four  hours  old. 
The  dose  is  0.02  c.c.,  or  one  capillary  drop;   inactivated  serums  are  used 
in  doses  of  0.08  c.c.,  or  four  capillary  drops.     Cerebrospinal  fluid  is  used 
unheated  in  doses  of  0.2  c.c.,  or  10  capillary  drops.     Sufficient  blood 
for  this  test  may  be  collected  in  a  Wright  capsule.     (See  p.  33.) 

6.  The  Test. — The  complement,  amboceptor,  antigen,  and  serums 
may  be  conveniently  measured  by  drops  from  a  capillary  pipet  (Fig. 
2).     In  placing  a  drop  the  pipet  should  be  held  uniformly  at  an  angle 
of  45  degrees,  or  else  the  size  of  the  drop  will  differ,  depending  on  whether 
the  pipet  is  held  vertically  or  horizontally. 

Arrange  four  pairs  of  small  test-tubes  (10  by  1  cm.)  in  a  rack  con- 
taining two  rows  cf  holes.  Into  each  of  the  tubes  on  the  front  row 


MODIFICATIONS    OF    THE    WASSERMANN    REACTION 


481 


place  five  drops  (0.1  c.c.)  of  antigen  emulsion  (alcoholic  solution,  1  part, 
with  saline  solution,  9  parts);  then  add  five  drops  (0.1  c.c.)  of  com- 
plement (40  per  cent.)  to  all  the  tubes.  Into  each  of  the  first  pair  of 
tubes  place  one  drop  (0.02  c.c.)  of  active  or  four  drops  (0.08  c.c.)  of 
inactivated  patient's  serum,  and  mark  the  front  tube  with  the  patient's 
name.  To  each  of  the  second  pair  of  tubes  add  an  equal  amount  of 
syphilitic  serum  known  to  give  a  positive  reaction  (positive  control),  and 
to  each  of  the  third  pair  add  normal  serum  known  to  give  a  negative  re- 
action (negative  control) .  Mark  the  tubes  in  the  front  row  of  each  pair 
respectively.  The  front  tube  of  the  fourth  pair  is  the  antigen  control, 
and  the  rear  tube  the  hemolytic  control,  and  each  should  be  so  labeled. 
Into  each  tube  place  1  c.c.  of  the  1  per  cent,  corpuscle  suspension  and  1 
c.c.  of  saline  solution,  making  the  total  volume  in  each  tube  about  2  c.c. 
Shake  each  tube  and  incubate  at  37°  C.  for  one  hour  (half  an  hour  in  the 
water-bath).  At  the  end  of  this  time  add  two  units  of  amboceptor  to 
each  tube,  shake  gently,  and  reincubate  for  two  hours  (one  hour  in  the 
water-bath).  During  this  time  the  tubes  should  be  shaken  gently 
once  or  twice  to  break  up  any  masses  of  agglutinated  corpuscles. 

The  following  chart,  after  Noguchi,  illustrates  the  various  steps  to 
be  taken  in  making  the  test  with  one  patient's  serum.  Of  course,  any 
number  of  serums  may  be  examined  with  the  same  controls  (Fig.  119). 

TABLE  16.— NOGUCHI  MODIFICATION  OF  THE  WASSERMANN 

REACTION 


SET  FOR 

POSITIVE                     NEGATIVE 

ANTIGEN  AND  HEM- 

DIAGNOSIS 

CONTROL  SET 

CONTROL  SET 

OLYTIC  CONTROLS 

2. 

4. 

6. 

8. 

^ 

, 

Unknown      se- 

Positive serum: 

Normal   serum  : 

Hemolytic    con- 

o 

c* 

i 

3 

c3 

rum  :  1  drop  l 

1  drop 

1  drop 

trol: 

o 

Complement:  5 
drops 

Complement:  5 
drops 

Complement:  5 
drops 

Complement:  5 
drops 

1 

I 

•* 

1  per  cent,  cor- 

1 per  cent,  cor- 

1 per  cent,  cor- 

1 per  cent,  cor- 

« 

5 

puscle      sus- 

puscle       sus- 

puscle       sus- 

puscle       sus- 

O, 

§ 

-^ 

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x  Four  drops  if  serum  is  inactivated. 


482  THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

At  the  end  of  the  second  incubation,  or  after  two  hours  more  at  room 
temperature,  the  tubes  are  inspected.  The  antigen  and  hemolytic 
system  controls,  as  well  as  all  the  rear  tubes  or  serum  controls,  should 
be  completely  hemolyzed.  The  first  tube  containing  a  known  syphilitic 
serum  shows  inhibition  of  hemolysis;  the  front  tube  containing  normal 
serum  is  completely  hemolyzed;  the  front  tube  containing  the  patient's 
serum  shows  complete  inhibition  of  hemolysis  (strong  positive),  varying 
degrees  of  inhibition  (moderately  or  weakly  positive),  or  is  completely 
hemolyzed  (negative) .  The  results  may  be  recorded  and  reported  after 
the  same  manner  described  on  p.  464. 

MODIFICATION  OF  CRAIG 

Craig1  employs  a  technic  differing  from  the  original  Wassermann 
reaction  in  the  use  of  a  human  hemolytic  system  in  place  of  a  sheep 
hemolytic  system,  and  in  a  proportional  reduction  in  the  quantities 
of  reagents  used.  Alcoholic  extract  of  syphilitic  liver  is  generally  em- 
ployed as  antigen,  although  cholesterinized  extracts  of  normal  heart 
muscle  have  been  found  equally  satisfactory.2  Vedder3  has  also  used 
Craig's  method  in  standardizing  the  Wassermann  reaction  for  use  in  the 
military  service,  and  it  is  widely  used  in  the  Army  with  satisfactory 
results. 

MODIFICATION  OF  BAUER 

Bauer  does  not  use  rabbit-sheep  amboceptor,  but  takes  advantage 
of  the  antisheep  amboceptor  normally  present  in  variable  amounts  in  a 
large  proportion  of  human  serums.  Although  this  test  is  quite  delicate, 
the  quantity  of  natural  amboceptor  in  human  serums  is  too  variable  a 
factor  to  be  depended  upon,  and  the  modification  is  not,  therefore,  in 
general  use. 

MODIFICATION  OF  HECHT-WFJNBERG-GRADWOHL 

In  conducting  the  syphilitic  reaction  Hecht4  utilizes  not  only  the 
natural  antisheep  amboceptor  in  human  serum  but  also  the  native 
hemolytic  complement.  The  serum  must  be  perfectly  fresh  and,  of 
course,  is  used  unheated.  This  modification  has  been  said  to  be  more 
delicate  than  the  Wassermann  reaction  because  none  of  the  syphilis  an- 
tibody is  destroyed  or  complementoids  produced,  as  presumably  will  oc- 
cur during  inactivation  (heating)  of  a  serum.  In  my  experience  this  test 

1  War  Department,  Bulletin  No.  3. 

2  Amer.  Jour.  Med.  Sci.,  1915,  cxlix,  41. 

3  War  Department,  Bulletin  No.  8. 

4  Wien.  klin.  Wchnschr.,  1909,  xxii,  265. 


MODIFICATIONS    OF   THE    WASSERMANN   REACTION  483 

has  proved  quite  delicate,  but  is  open  to  the  same  error  that  may  occur 
whenever  an  active  serum  is  used  with  a  crude  alcoholic  organic  extract 
as  antigen — i.  e.,  the  appearance  of  false  positive  or  proteotropic  reac- 
tions. As  with  the  Noguchi  reaction,  using  active  serum,  a  negative 
Hecht-Weinberg  test  has  considerable  diagnostic  value  in  excluding 
syphilis;  a  positive  reaction  must  be,  however,  carefully  controlled. 
In  performing  the  test  I  always  use  an  extract  of  acetone-insoluble 
lipoids  as  antigen. 

Since  in  the  original  Hecht-Weinberg1  test  there  is  no  way  of  deter- 
mining beforehand  the  amount  of  sheep  corpuscles  a  serum  may  hemo- 
lyze,  Gradwohl2  has  modified  the  technic  so  that  the  hemolytic  index 
of  each  serum  is  determined  before  the  corpuscles  are  added  to  the 
main  tubes.  I  conduct  this  test  as  follows  :8  Nine  small  sterile  test-tubes 
(10x1  cm.)  are  arranged  for  each  serum  and  properly  labeled;  into  each 
is  placed  0.1  c.c.  of  fresh  unheated  serum.  To  the  first  five  tubes  are 
added  respectively  0.1, 0.2, 0.3, 0.4,  and  0.5  c.c.  of  a  5  per  cent,  suspension 
of  washed  sheep  cells;  to  the  sixth,  seventh,  and  eighth  tubes  are  added 
1,  2,  and  3  units  of  antigen  respectively,  as  determined  by  titration. 
The  last  or  ninth  tube  serves  as  the  serum  control.  Sufficient  normal 
salt  solution  is  added  to  each  tube  to  bring  the  total  volume  to  1  c.c. 

After  one  hour's  incubation  at  37°  C.  the  hemolytic  index  of  each  serum 
is  read  in  the  first  five  tubes  of  each  series  (that  is,  the  largest  dose  of 
corpuscles  just  completely  hemolyzed  by  each  serum)  and  one-half  the 
indicated  doses  of  corpuscles  added  to  the  remaining  four  tubes  of  each 
series.  After  re-incubation  for  one-half  to  one  hour,  according  to  the 
hemolysis  of  the  controls,  the  results  are  read  and  recorded  as  in  the 
Wassermann  reaction. 

The  antigen  titrations  are  very  important  because  the  activity  of 
human  serum  is  quite  sensitive  to  the  inactivating  influence  of  tissue 
extracts.  No  antigen  should  be  employed  in  this  test  without  pre- 
liminary titration  with  human  serum  to  determine  its  anticomplementary 
and  antigenic  units.  The  anticomplementary  titration  is  conducted  by 
placing  in  a  series  of  eight  test-tubes  the  following  amounts  of  antigen 
of  acetone-insoluble  lipoids  diluted  1  : 100:  0.2,  0.3,  0.4,  0.6,  0.8,  1.0, 
1.2, 1.5  c.c.,  and  0.1  c.c.  of  the  fresh  and  mixed  sera  of  two  non-syphilitic 
persons;  normal  salt  solution  is  added  to  2  c.c.  and  the  tubes  incubated 
for  an  hour  at  37°  C.,  when  one-half  the  index  of  cells  for  the  serum 
is  added  to  each  tube.  After  a  second  incubation  of  an  hour  the  anti- 

1  Wien.  klin.  Wchnschr.,  1908,  21,  1742;  ibid.,  1907,  22,  265;  find.,  1909,  22,  338. 

2  Jour.  Amer.  Med.  Assoc.,  1914,  63,  240.  3  Jour.  Immunol.,  1916,  2,  23. 


484  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

complementary  unit  is  read  as  the  smallest  dose  of  antigen  producing 
inhibition  of  hemolysis.  The  antigenic  titration  is  conducted  in  the  same 
manner  with  the  serum  of  one  or  two  syphilitic  persons  and  the  follow- 
ing doses  of  antigen  (1  :  100) :  0.05,  0.1,  0.15,  0.2,  0.25,  and  0.3  c.c.  The 
antigenic  unit  is  the  smallest  amount  giving  complete  inhibition  of 
hemolysis. 

MODIFICATION  OF  STERN 

Margaretta  Stern  devised  a  modification  of  the  Wassermann  reac- 
tion, using  fresh  active  serum  and  the  patient's  complement,  and  over- 
coming non-specific  reactions  by  using  f  to  -|-  of  the  usual  dose  of 
extract,  and  three  or  four  times  the  amboceptor  unit.  This  method  is 
open  to  defects  inherent  in  the  use  of  variable  amounts  of  complement 
and  excessive  amounts  of  hemolytic  amboceptor,  which  makes  it  im- 
possible to  test  a  specimen  a  few  days  after  it  has  been  collected. 

MODIFICATION  OF  TCHERNOGUBOU 

Tchernogubou  proposed  an  antihuman  hemolytic  system  with  active 
serum.  Blood  is  collected  in  sodium  citrate,  and  therefore  contains 
erythrocytes,  complement,  and  syphilis  antibody  if  the  patient  is  luetic. 
Antigen  is  added,  and  after  sufficient  time  has  elapsed  for  fixation  of 
complement  to  take  place,  antihuman  amboceptor  is  added  to  test  for 
free  complement.  There  are  many  objections  to  this  method,  the  chief 
ones  being  the  variable  amount  of  complement  present  in  human  serum, 
the  large  amount  of  hemolytic  amboceptor  required,  the  absence  of  a 
suitable  control  on  the  antigen,  and  the  fact  that  old  blood  is  entirely 
unsuited  for  making  the  test. 

Tchernogubou  has  also  proposed  a  system  in  which  the  natural 
amboceptor  and  complement  of  human  serum  are  utilized  against 
guinea-pig  corpuscles.  These  factors  are  so  variable  that  this  modified 
test  has  been  largely  abandoned. 

MODIFICATION  OF  DETRE  AND  BREZOVSKY 

In  this  modification  an  antihorse  hemolytic  system  is  used  with 
rabbit's  complement.  The  chief  objections  are  the  variations  in  the 
activity  and  fixability  of  rabbit  complement,  and  the  difficulty  of  ob- 
taining horse  blood.  In  addition  to  these,  this  method  possesses  no 
advantages  over  Wassermann's  antisheep  system. 

MODIFICATION  OF  BROWNING  AND  MACKENZIE 

These  investigators  use  an  antiox  hemolytic  system,  and  have 
modified  the  original  Wassermann  technic  so  as  to  make  the  method  an 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  485 

accurate  quantitative  test.  (See  p.  470.)  They  claim  that  with  their 
modified  technic  the  results  secured  are  practically  the  same  with  the 
antiox  and  antisheep  systems,  although  the  human  serums  contain 
much  less  natural  antiox  amboceptor  and,  theoretically,  therefore  this 
system  is  better. 

MODIFICATION  OF  VON  DUNGERN 

More  recently  von  Dungern  has  proposed  a  modification  similar  to 
that  of  Noguchi.  Like  Tchernogubou,  he  uses  active  serum  only,  and 
utilizes  the  patient's  own  blood-cells.  The  blood  is  defibrinated  and 
used  in  doses  of  0. 1  c.c.  Complement  in  the  human  blood  is  disregarded, 
and  is  furnished  by  guinea-pig  serum  in  dried-paper  form.  Alcoholic 
extract  of  syphilitic  liver  is  used  as  antigen,  and  this  opens  up  an  avenue 
for  false  positive  or  proteotropic  reactions  to  creep  in.  Antihuman 
amboceptor  is  prepared  by  immunizing  goats,  but,  as  Noguchi  has  shown, 
this  amboceptor  gives  a  much  weaker  hemolytic  reaction  than  that  de- 
rived from  the  rabbit.  Von  Dungern  generally  omits  the  important 
control  with  known  syphilitic  serum;  there  is  no  direct  control  on  the 
antigen,  and  old  bloods  cannot  be  used. 

THE  WASSERMANN  REACTION  IN  THE  VARIOUS  STAGES  OF  SYPHILIS 
1.  In  Primary  Syphilis. — As  would  be  expected,  a  certain  degree  of 
tissue  change  most  occur  before  syphilis  reagin  appears  in  the  blood. 
Even  with  the  best  technic  there  is  a  limit  to  the  sensitiveness  of  the 
Wassermann  reaction,  so  that  while  the  reagin  may  be  produced  at  the 
very  onset  of  an  infection,  time  and  further  tissue  changes  are  required 
before  sufficient  reagin  is  produced  to  yield  a  complement-fixation 
reaction. 

Since,  therefore,  the  results  of  the  Wassermann  reaction  in  primary 
syphilis  are  dependent  upon  the  virulence  of  the  infection,  the  time  at 
which  the  reaction  is  made,  and  the  delicacy  of  the  technic,  it  is  not 
surprising  that  the  results  of  different  investigators  vary  in  the  propor- 
tion of  positive  reactions  obtained.  While  positive  reactions  have  been 
said  to  have  been  secured  before  the  appearance  of  the  initial  lesion, 
these  are  rare,  and  there  is  always  the  likelihood  that  an  earlier  infection 
was  overlooked.  A  careful  review  of  our  own  work  and  the  literature 
upon  this  subject  establishes  the  following: 

(a)  A  positive  reaction  may  be  secured  during  the  first  week  after 
the  appearance  of  the  chancre.  Craig  has  reported  a  positive  reaction 
occurring  five  days  after  the  appearance  of  the  initial  lesion.  Levaditi, 
Laroche  and  Yamanouchi,  and  others  have  recorded  many  positive 


486  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

reactions  occurring  in  ten  days  or  more  after  the  chancre  made  its  ap- 
pearance. 

(6)  In  general,  in  primary  syphilis  the  Wassermann  reaction  will  be 
positive  in  about  80  to  90  per  cent,  of  cases;  where  cholesterinized  ex- 
tracts are  used  as  antigens,  or  with  the  Noguchi  system,  using  active 
serum,  the  reactions  are  secured  earlier  and  in  a  larger  percentage  of 
cases.  Craig1  has  reported  34  per  cent,  positive  reactions  during  the 
first  week  after  the  appearance  of  the  chancre;  57  per  cent,  during  the 
second  week;  67  per  cent,  during  the  third  week;  76  per  cent,  during  the 
fourth  week,  and  80  per  cent,  during  the  fifth  week. 

(c)  It  is  generally  agreed  that  a  diagnosis  should  be  made  as  early  as 
possible,  and  vigorous  treatment  instituted.     A  Wassermann  reaction 
may  be  performed,  and  if  it  shows  a  positive  result,  this  indicates  the 
presence  of  syphilis,  even  if  the  lesion  under  suspicion  is  not  specific,  the 
reaction  being  due  to  a  previous  infection.     A  negative  reaction,  how- 
ever, does  not  exclude  syphilis,  and  if  it  is  at  all  possible,  a  microscopic 
examination,  using  the  dark-ground  illuminator,  should  be  made  for 
the  treponema.     In  primary  syphilis  a  microscopic  examination  of  the 
secretions  of  the  lesion  by  a  competent  person  is  usually  more  valuable 
than  the  serum  test;    as  a  general  rule,  both  examinations  should  be 
made,  especially  with  patients  in  whom  the  chancre  is  almost  healed  or 
atypical. 

(d)  The  cerebrospinal  fluid  of  persons  in  the  primary  stage  of  syphilis 
has  always  reacted  negatively  (Boas). 

2.  In  Secondary  Syphilis. — It  is  in  untreated  cases  of  secondary 
syphilis  that  the  remarkable  specificity  of  the  Wassermann  reaction 
is  so  well  demonstrated.  The  initial  lesion  may  have  been  inconspicuous 
and  hence  have  been  overlooked,  and  the  secondary  lesions  may  be 
quite  mild  and  inconclusive;  in  either  case  the  Wassermann  reaction 
will  usually  establish  the  diagnosis. 

(a)  In  untreated  secondary  syphilis  the  reaction  is  positive  in  from 
92  to  100  per  cent,  of  cases.  In  the  examination  of  437  serums  from 
untreated  cases  Boas  has  never  had  a  negative  reaction,  and  my  own 
experience  has  been  the  same.  Craig  reports  96  per  cent,  positive  re- 
actions. 

(6)  With  the  serums  of  patients  who  have  received  some  treatment 
the  percentage  of  positive  reactions  will  be  slightly  lower.  Of  310  such 
cases  examined  by  Boas,  97.6  reacted  positively.  The  influence  of 
treatment  upon  the  reaction  is  to  be  remembered,  and  a  single  negative 
reaction  does  not  by  any  means  exclude  the  possibility  of  syphilis. 
1  Amer.  Jour.  Med.  Sci.,  1915,  cxlix,  41. 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  487 

(c)  The  intensity  of  the  reaction  does  not  bear  any  direct  relation 
to  the  severity  of  the  infection:    a  mild  infection  with  indefinite  signs 
may  react  quite  strongly  and  absorb  a  large  number  of  units  of  comple- 
ment, whereas  a  severe  case  may  react  quite  mildly. 

(d)  In  secondary  syphilis  without  cerebral  symptoms  the  cerebro-'/ 
spinal  fluid  is  practically  always  negative  (Plaut,  Boas  and  Lind); 
conversely,  cases  showing  cerebral  involvement  usually  react  positively. 
More  recent  work  has  shown  that  the  cerebrospinal  system  is  involved 
early  and  in  a  relatively  large  number  of  cases  (Craig  and  Collins1). 
Udo  J.  Wile  has  found  that  about  30  per  cent,  of  secondary  syphilitics 
give  a  positive  reaction  with  cerebrospinal  fluid. 

3.  In  Tertiary  Syphilis. — It  is  probably  in  tertiary  syphilis  that  the  / 
Wassermann  reaction  has  its  greatest  value.  Lues  is  so  diverse  in 
character,  and  may  be  responsible  for  so  many  diverse  clinical  conditions, 
that  the  reaction  has  become  well-nigh  indispensable  as  a  diagnostic 
aid.  There  is  no  limit  to  the  time  following  infection  in  which  positive 
reactions  may  not  be  found. 

(a)  In  cases  of  untreated  and  active  tertiary  syphilis  the  reaction  is 
positive  in  about  96  per  cent,  of  cases. 

(6)  In  cases  receiving  more  or  less  antispecific  treatment  the  reac- 
tions are  positive  in  about  75  per  cent.  In  general,  therefore,  a  positive 
reaction  in  tertiary  syphilis  may  be  expected  in  from  80  to  85  per  cent, 
of  cases. 

(c)  In  a  large  percentage  of  cases  of  syphilitic  aortitis,  aortic  aneu-  " 
rysm,  aortic  insufficiency,  gummas  of  various  organs,  etc.,  the  reaction 
is  positive  and  possesses  great  diagnostic  value. 

(d)  The  Wassermann  reaction  has  been  especially  valuable  in  the 
study  of  the  so-called  parasyphilitic  diseases.     In  general  paralysis  or 
paralytic  dementia  the  serum  reacts  positively  in  about  100  per  cent,  of 
cases,  and  the  cerebrospinal  fluid  reacts  positively  in  about  92  per  cent. 
The  final  and  conclusive  proof  of  the  syphilitic  nature  of  this  disease 
has  been  furnished  by  Noguchi  and  Moore,  who  found  the  Treponema 
pallidum  in  sections  of  the  brain.     In  certain  cases  of  general  paralysis 
the  blood-serum  may  react  negatively,  whereas  with  the  cerebrospinal 
fluid  the  reaction  is  positive. 

The  fact  that  the  blood-serum  of  a  patient  with  a  nervous  disease  • 

reacts  positively  does  not  necessarily  indicate  that  the  nervous  disease 

is  of  syphilitic  origin,  as  the  reaction  may  be  due  to  specific  infection  of 

some  other  structure;    if,  however,  the  cerebrospinal  fluid  also  reacts 

1  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1955. 


488  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

positively,  then  it  is  almost  certain  that  syphilitic  infection  of  the 
central  nervous  system  is  present. 

In  untreated  and  active  cases  of  tabes  dorsalis  the  blood-serum  reacts 
positively  in  from  96  to  100  per  cent,  of  cases.  In  treated  cases  the 
number  of  positive  reactions  drops  to  about  40  to  50  per  cent;  in  general, 
therefore,  a  positive  reaction  with  the  serums  of  tabetics  may  be  ex- 
pected in  73  per  cent,  of  cases.  With  the  cerebrospinal  fluid  the  per- 
centage of  positive  reactions  is  somewhat  lower,  being  about  60  per 
cent.  The  positive  Wassermann  reaction  is  less  constant  in  locomotor 
ataxia  than  in  general  paralysis,  due  probably  to  the  fact  that  the  former 
is  more  chronic  and  that  intercurrent  periods  of  arrest  are  more  prone 
to  occur. 

In  cerebral  syphilis  the  blood-serum,  and  particularly  the  cerebro- 
spinal fluid,  will  react  positively  less  frequently  than  in  general  paralysis. 
In  some  instances  a  positive  reaction  is  found  with  the  cerebrospinal 
fluid  and  not  with  the  serum,  a  matter  difficult  to  explain  and  believed 
to  be  due  to  the  confining  of  the  reacting  substances  in  the  subarachnoid 
space.  On  the  other  hand,  the  lesions  are  probably  not  brought  in 
direct  contact  with  the  spinal  fluid. 

There  is  much  evidence  to  indicate  that  localization  of  syphilis  in  the 
nervous  system  is  dependent  upon  a  particular  strain  of  Treponema 
pallidum;  other  strains  appear  to  possess  a  special  affinity  for  the 
visceral  organs,  bones,  etc. 

(e)  In  tertiary  syphilis  not  accompanied  by  lesions  of  the  central 
nervous  system  the  Wassermann  reaction  with  cerebrospinal  fluid  may 
be  positive  in  a  relatively  large  percentage  of  cases. 

4.  In  Latent  Syphilis. — In  cases  of  latent  syphilis  the  Wassermann 
reaction  may  constitute  the  only  evidence  of  the  existence  of  the  disease, 
and  prompt  institution  of  treatment  may  prevent  the  development  of 
tertiary  lesions,  which  are  so  likely  to  follow.  When  the  spirochetes 
are  few  in  number  and  are  dormant,  there  is  little  tissue  destruction  or 
alteration,  and,  as  a  result,  so  little  reagin  is  frequently  present  in  the 
body-fluids  that  the  Wassermann  reaction  will  fail  to  detect  the  disease. 

(a)  In  363  cases  of  early  latent  syphilis,  or  those  included  within  a 
period  of  three  years  after  infection,  Boas  found  positive  reactions  in 
about  40  per  cent. ;  in  latent  cases  of  long  standing,  or  in  those  following 
manifest  tertiary  lesions,  the  same  investigator  found  22  per  cent,  of 
positive  reactions  among  those  who  had  received  proper  treatment;  of 
those  receiving  indifferent  treatment,  74  per  cent,  reacted  positively, 
giving  a  general  average  of  about  48  per  cent.  Craig  has  found  67  per 
cent,  positive  reactions  in  latent  syphilis. 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  489 

(6)  The  reaction  with  cerebrospinal  fluid  depends  upon  whether  or 
not  the  central  nervous  system  is  involved  in  the  syphilitic  process.  Of 
104  latent  cases  of  syphilis  in  whom  the  spinal  fluid  was  examined  by 
Altman  and  Dreyfus,  positive  reactions  were  found  in  about  10  per  cent. 

5.  Congenital  Syphilis. — The  Wassermann  reaction  has  thrown 
considerable  light  upon  the  subject  of  congenital  syphilis.  While,  in 
general,  the  majority  of  cases  react  positively,  the  results  are  largely 
dependent  upon  the  time  when  the  examinations  are  made,  a  fact  brought 
out  by  the  highly  instructive  and  systematic  investigations  of  Boas  and 
Thomsen.  These  investigators  divided  their  cases  into  three  groups: 
(1)  Newly  born  children  and  their  mothers;  (2)  two-year-old  children; 
(3)  older  children  with  congenital  syphilis. 

(a)  Of  88  children  born  of  syphilitic  mothers  and  examined  at  birth, 
the  reaction  was  positive  in  31  and  negative  in  57  cases.  Of  the  31 
positive  cases,  4  showed  no  symptoms  of  syphilis  for  a  period  of  observa- 
tion covering  from  three  to  nine  months,  and  it  is  possible  that  the 
syphilis  reagin,  and  not  the  spirochetes,  from  the  blood  of  the  mother, 
passed  into  the  circulation  of  the  child;  on  the  other  hand,  all  four  cases 
may  have  been  examples  of  retarded  congenital  syphilis.  The  remaining 
27  cases  either  developed  symptoms  of  syphilis  or  died  later  with  syphil- 
itic manifestations  in  various  organs. 

Of  the  57  children  reacting  negatively  at  birth,  42  showed  no  symp- 
toms of  syphilis  during  a  period  of  three  months  of  observation;  2  died 
with  evidences  of  syphilis  in  the  internal  organs;  13  developed  symptoms 
after  birth  and  gave  positive  reactions. 

It  may  therefore  be  stated  that  a  Wassermann  reaction  of  the  mother 
and  of  the  child  at  the  time  of  birth  in  cases  where  syphilis  of  the  mother 
is  suspected  has  considerable  prognostic  value.  A  large  majority  of 
children  reacting  positively  develop  symptoms  of  syphilis;  on  the  other 
hand,  the  majority  reacting  negatively  remain  healthy.  While  an 
examination  of  the  mother  alone  does  not  warrant  an  absolutely  definite 
prognosis  for  the  child,  in  general  it  may  be  said  that  a  positive  reaction 
does  not  constitute  a  favorable  prognostic  sign  for  the  child. 

(6)  The  Wassermann  reaction  has  also  shed  new  light  upon  the 
interpretation  of  Colics'  law.  Since  the  "  apparently  healthy  mother 
of  a  syphilitic  child  could  suckle  the  child  without  being  infected,  whereas 
the  child  is  capable  of  giving  syphilis  to  others,"  the  most  logical  con- 
clusion to  draw  is  that  the  mother  was  gradually  immunized  against 
syphilis  during  pregnancy,  whereas  we  now  know  that  the  majority  of 
mothers  show  positive  serum  reactions  and  are  really  latent  syphilitics; 
in  not  a  few  such  instances  tertiary  lesions  have  developed  at  a  later  date. 


490  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

It  is  possible,  however,  for  a  syphilitic  mother  showing  a  positive 
Wassermann  reaction  to  give  birth  to  a  healthy  child.  Of  46  mothers 
whose  children  showed  no  evidences  of  syphilis  over  a  period  of  ob- 
servation of  three  months,  17  reacted  positively.  Of  81  mothers  giving 
birth  to  syphilitic  children,  61  reacted  positively,  and  many  of  these 
would  naturally,  in  former  years,  have  been  regarded  as  examples  of 
Colles'  immunity  and  considered  free  of  syphilis.  In  many  instances 
the  apparently,  healthy  child  of  a  syphilitic  mother  that  could  not  be 
infected  by  the  mother  (Profeta's  law)  has  been  shown  by  the  Wasser- 
mann reaction  to  be  in  reality  a  case  of  retarded  congenital  syphilis, 
and  that  such  children  are  not  immunized,  during  intra-uterine  life, 
either  passively  or  by  means  of  pallidum  toxins,  against  syphilis,  as  has 
been  so  generally  believed  in  past  years.  In  other  words,  there  appears 
to  be  no  lasting  passive  immunity  in  syphilis;  it  is  doubtful  if  the  toxins 
of  pallidum  can  pass  between  mother  and  child  and  immunize  one  or  the 
other  without  actual  infection  with  the  spirochetes  themselves  taking 
place;  that  most  examples  of  so-called  immunity  in  syphilis  in  both  the 
mother  (Colles'  law)  and  the  child  (Profeta's  law)  are  due  to  the  actual 
presence  of  pallidum  in  the  tissues  and  are  really  latent  infections. 

(c)  In  manifest  untreated  congenital  syphilis  of  children  one  year  or 
over  in  age  the  Wassermann  reaction  is  positive  in  from  97  to  100  per 
cent,  of  cases.     The  clinical  manifestations  may  be  quite  varied  and 
clinically  ill  defined,  so  that  the  serum  reaction  possesses  considerable 
diagnostic  value.     In  most  instances  the  reactions  are  quite  strong,  and 
while  active  treatment  may  improve  local  lesions,  it  is  very  difficult, 
indeed,  to  secure  negative  reactions. 

(d)  In  congenital  mental  deficiency  and  epilepsy  the  Wassermann 
reaction  shows  that  syphilis  pla3rs  a  larger  part  in  the  etiology  of  this 
condition  than  is  generally  supposed.    A  riot  inconsiderable  proportion 
of  cases  are  of  infectious  origin,  and  that  infection  is  syphilis.    In  Little's 
disease,  which  is  regarded  as  due  to  rneningeal  hemorrhage  incidental 
to  injury  received  during  labor,  the  serum  reactions  have  shown  that  not 
infrequently  the  hemorrhage  has  a  syphilitic  origin. 

THE  SPECIFICITY  OF  THE  WASSERMANN  REACTION 

The  highly  specific  nature  of  the  syphilis  reaction  has  been  proved 

by  very  extensive  investigations  with  the  serums  of  normal  persons  and 

'of  persons  afflicted  with  diseases  other  than  syphilis.     Unfortunately, 

the  reaction  is  beset  by  so  many  technical  errors  that  a  review  of  the 

literature,  and  especially  of  the  early  literature,  shows  results  that  are 


MODIFICATIONS    OF   THE  WASSERMANN   REACTION  491 

quite  confusing  and  contradictory.  Following  the  original  communica- 
tions of  Wassermann  and  Detre,  and  especially  after  it  was  demonstrated 
that  the  antigen  need  not  be  biologically  specific,  the  subject  was  ex- 
tensively investigated  by  various  observers,  who  reported  securing 
positive  reactions  in  many  different  diseases,  results  that  we  now  know 
must  have  been  due  largely  to  technical  errors.  At  present  it  is  known 
that  positive  Wassermann  reactions  may  occur  in  a  few  diseases  other 
than  syphilis,  but  not  to  the  extent  that  earlier  investigators  would  have 
us  believe.  In  most  of  the  diseases  yielding  positive  reactions  the 
clinical  symptoms  are  so  marked  that  they  may  readily  be  differentiated 
from  syphilis,  and  accordingly  the  Wassermann  reaction  is  of  unequaled 
and  incalculable  diagnostic  value. 

Positive  reactions  have  been  reported  mframbesia  (yaws),  in  which 
the  causal  microorganism,  the  Spirochaeta  pertenuis,  is  morphologically 
almost  indistinguishable  from  Spirochaeta  pallidum.  In  leprosy  of  the 
tuberous  type  positive  reactions  are  frequently  found,  but  cases  of 
anesthetic  leprosy  usually  react  negatively.  Positive  reactions  have 
been  reported  in  cases  of  malaria  during  the  febrile  stage,  when  para- 
sites are  present,  although  the  majority  of  cases  react  negatively.  In 
my  own  series  of  11  cases  all  the  reactions  were  negative.  Positive 
reactions  have  also  been  found  in  some  cases  of  relapsing  fever. 

In  scarlet  fever  the  Wassermann  reaction  is  uniformly  negative. 
Owing  to  the  original  communication  of  Much  and  Eichelberg,  however, 
in  the  minds  of  many  this  disease  is  prominently  associated  with  a 
positive  reaction.  While  it  is  true  that  a  positive  reaction  is  very  rarely 
found,  it  is  almost  impossible  entirely  to  exclude  a  diagnosis  of  con- 
genital lues,  at  least  in  some  of  these  cases.  In  my  own  series  of  250 
cases  examined  by  the  Wassermann  and  Noguchi  methods,  with  antigens 
of  alcoholic  extract  of  syphilitic  liver  and  acetone-insoluble  lipoids,  the 
reactions  were  positive  in  5  cases,  or  2  per  cent.  Similar  results  have 
been  secured  by  Boas,  Browning  and  Mackenzie,  and  others,  so  that  it 
may  be  said  that  the  reaction  in  scarlet  fever  is  uniformly  negative.  It  is 
also  to  be  remembered  that  occasionally,  or  in  about  1  to  2  per  cent,  of 
cases,  a  positive  reaction  may  follow  ether  or  chloroform  anesthesia,  but 
that  this  will  later  disappear.  In  pellagra  Fox,  and  later  Bass,  have 
found  occasional  positive  reactions.  Craig  and  Nickols  have  reported 
that  blood  drawn  from  Wassermann  positive  persons  immediately  after 
an  acute  alcoholic  debauch  may  yield  a  negative  result. 

Normal  cerebrospinal  fluid  or  the  fluid  from  persons  with  ordinary 
non-syphilitic  diseases  reacts  negatively.  Positive  reactions  have  been 
reported  in  leprosy  and  in  frambesia. 


492          THE  TECHNIC  OF  COMPLEMENT-FIXATION  REACTIONS 

THE  EFFECT  OF  TREATMENT  UPON  THE  WASSERMANN  REACTION 
Citron  originally  observed  that  during  the  mercurial  treatment  of 
syphilis  the  Wassermann  reaction  gradually  became  weaker,  and  finally 
disappeared.  He  also  found  that  treatment  was  best  governed  by  the 
serum  reaction,  and  that  it  should  be  persisted  in  until  a  negative  re- 
action was  secured.  His  observations  have  in  the  main  been  abundantly 
confirmed  by  various  observers  the  world  over,  although  the  extensive 
series  of  observations  now  on  record  have  given  us  a  fuller  understanding 
of  its  principles. 

The  Wassermann  reaction  is  the  most  constant  and  delicate  single 
symptom  of  syphilis,  and  whenever  a  serum  is  found  to  react  positively, 
antisyphilitic  treatment  is  indicated,  and  should  be  persisted  in  until 
the  reaction  becomes  negative  and  remains  so  for  a  sufficiently  pro- 
longed period  of  observation.  It  is  now  quite  generally  believed  that 
a  persistently  positive  reaction  indicates  the  presence  of  living  spiro- 
chetes,  and  that  treatment  should  be  continued  until  the  blood  reacts 
negatively.  The  reports  of  observers  from  all  parts  of  the  world  indi- 
cate quite  clearly  and  conclusively  that  the  schematic,  symptomatic, 
intermittent,  and  hard  and  fast  rules  of  treatment  of  former  days  are 
not  sufficient.  They  would  also  tend  to  show  that  the  Wassermann 
reaction  is  the  most  delicate  symptom  and  the  last  to  disappear,  and 
that  treatment  should  be  continued  until  this  reaction  disappears 
entirely  and  permanently.  It  has  been  abundantly  proved,  however,  that 
in  syphilis  a  single  negative  reaction  is  not  sufficient  or  definite  evidence 
that  a  cure  has  been  effected,  for  the  disease  may  recur  after  treatment  is 
discontinued,  at  least  to  the  extent  that  the  Wassermann  reaction  reappears, 
followed  by  clinical  manifestations.  It  is  necessary,  therefore,  that  sue- 
cessive  examinations  be  made  during  a  period  of  at  least  two  years,  and  off 
and  on  during  the  remainder  of  life.  Recent  work  indicates  that  certain 
strains  of  Spirochseta  pallida  have  an  apparent  selective  affinity  for  the 
tissues  of  the  central  nervous  system;  the  Wassermann  reaction  with 
blood-serum  may  be  negative,  whereas  with  the  cerebrospinal  fluid  it 
may  be  positive.  In  cases,  therefore,  of  tertiary  syphilis,  at  least,  it  is 
advisable  to  examine  the  spinal  fluid  and  continue  treatment  in  case  it 
shows  a  positive  Wassermann  reaction. 

It  should  be  the  object  of  treatment,  in  every  case,  not  only  to  dis- 
sipate the  external  and  obvious  lesions  of  the  disease,  but  to  produce  a 
condition  of  the  blood  in  which  the  Wassermann  reaction  is  permanently 
negative.  It  is  quite  generally  agreed  that  the  older  methods  of  treat- 
ment, consisting  of  the  administration  of  mercury  and  the  iodids  over 
fixed  and  arbitrary  periods  of  time,  or  until  all  manifest  symptoms  have 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  493 

disappeared,  are  insufficient,  and  that  the  criteria  by  which  the  effects  of 
treatment  can  best  be  judged  are:  (1)  Continued  absence  of  symptoms, 
and  (2)  permanent  negative  Wassermann  reactions. 

It  is  to  be  remembered,  therefore,  that  while  a  single  negative 
reaction  is  a  satisfactory  indication  of  the  progress  of  treatment,  it  does 
not  signify  that  a  permanent  cure  has  been  effected.  The  Wassermann 
reaction  cannot  be  regarded  as  sufficiently  delicate  to  indicate  that  a 
single  negative  reaction  means  that  a  patient  is  totally  free  from  all 
spirochetes,  for  in  some  instances  the  reaction  and  the  clinical  symptoms 
may  recur  after  the  treatment  has  been  suspended,  but  the  reaction  is 
the  first  symptom  to  reappear  and  the  earliest  indication  of  an  impending 
lesion.  For  all  practical  purposes  the  occurrence  of  a  negative  reaction 
after  treatment  indicates  either  complete  destruction  of  all  the  spiro- 
chetes, or  at  least  that  the  parasites  are  being  held  in  abeyance  and 
rendered  potentially  harmless. 

It  is,  accordingly,  reasonable  to  regard  the  Wassermann  reaction  as 
the  most  delicate  indicator  of  generalized  spirochetal  infection  or  the 
assumption  of  spirochetal  activity.  A  positive  reaction  indicates  that 
serious  effects  and  gross  local  lesions  are  likely  to  occur  at  any  time,  and 
that  treatment  should  be  continued.  For  all  practical  purposes  a  con- 
tinued absence  of  symptoms  and  a  permanently  negative  reaction  are 
strong  presumptive  evidences  that  a  cure  has  been  effected. 

The  serum  should  be  tested  every  six  months  during  the  treatment, 
and  at  periods  of  at  least  six  months  to  a  year  after  treatment  has  been 
discontinued  for  several  years.  Persistently  positive  reactions  during 
treatment  would  indicate  that  more  active  measures  or  a  change  in 
therapy  are  needed.  The  occurrence  of  a  positive  reaction  after  treat- 
ment has  been  discontinued  is  an  indication  for  its  resumption. 

For  a  control  on  treatment  the  Wassermann  reaction  should  be  made 
as  delicate  as  possible,  for  while  more  prolonged  treatment  may  be  some- 
what irksome  to  the  patient,  it  is  clearly  indicated  as  a  preventive  of 
serious  after-effects,  especially  of  involvement  of  the  central  nervous 
system.  It  is  in  this  branch  of  the  work  I  have  found  that  the  use  of 
sensitive  cholesterinized  extracts  as  antigens  in  making  the  Wassermann 
reaction  or  the  Noguchi  modification  with  the  use  of  active  serum,  of 
great  value  as  the  most  delicate  indicators. 

One  fact  is  to  be  clearly  emphasized,  namely,  that  the  earlier  energetic 
treatment  is  begun,  the  more  likely  it  is  that  a  permanent  cure  will  be  effected. 
Energetic  treatment  with  mercurials  or  salvarsan,  or,  better,  with  a 
combination  of  both,  begun  early  and  continued  long,  will  in  the  majority 
of  cases  restore  the  serum  to  its  normal  condition.  In  general,  the 


494  THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

greater  the  interval  of  time  allowed  to  elapse  between  infection  and  in- 
stitution of  treatment,  the  more  difficult  it  is  to  restore  the  serum  to 
normal.  Tertiary  cases  are  cured  only  as  the  result  of  most  persistent 
treatment,  and  not  infrequently  in  congenital  syphilis,  locomotor  ataxia, 
and  general  paralysis  all  one  can  hope  to  accomplish  is  to  check  the 
progress  of  the  disease.  The  most  favorable  cases  are  those  in  which 
early  diagnosis  is  made  possible  by  clinical  manifestations,  preferably 
confirmed  by  a  demonstration  of  pallidum,  and  in  which  treatment  is 
undertaken  before  the  serum  has  begun  to  react  positively,  and  in  which  the 
reaction  remains  negative  throughout. 

Treatment  will,  however,  at  least  influence  the  Wassermann  reaction 
in  practically  all  stages  of  syphilis.  In  a  series  of  435  cases  of  syphilis 
in  all  stages  reported  by  Boas,  a  negative  Wassermann  reaction  was 
secured  in  no  less  than  80  per  cent.,  and  all  but  one  of  the  remaining  cases 
showed  a  weaker  reaction.  The  figures  of  different  observers  are  not 
all  so  favorable  as  these,  a  factor  dependent  to  some  extent,  at  least, 
upon  differences  in  the  technic  of  the  reaction.  In  general,  however, 
Boas'  observations  have  been  confirmed  by  other  competent  workers. 

The  effect  of  any  treatment  is  greatly  influenced  by  the  individuality 
of  the  host,  certain  persons  possessing  tissues  more  amenable  to  the 
effects  of  the  therapeutic  agent  than  those  of  others.  The  therapeutic 
effect  is  also  dependent  upon  the  virulence  of  the  parasite  and  the 
apparent  selective  affinity  of  certain  strains  of  pallidum  for  particular 
organs,  and  upon  the  method  of  treatment  selected. 

The  influence  of  salvarsan  and  neosalvarsan  as  agents  in  the  treat- 
ment of  syphilis  is  considered  in  more  detail  in  the  chapter  on  Chemo- 
therapy. My  experience  has  shown  that  the  earlier  belief  in  the  com- 
plete sterilization  of  the  human  patient  by  a  single  dose  was  generally 
unfounded,  and  that  repeated  smaller  doses  of  the  drug,  used  in  con- 
junction with  mercurials,  are  necessary.  Potassium  iodid  alone  may 
favorably  influence  the  clinical  symptoms  and  weaken  the  Wassermann 
reaction  in  a  small  percentage  of  cases,  and  the  same  result  has  been  ob- 
served with  such  arsenical  preparations  as  Fowler's  solution,  atoxyl, 
arsacetin,  and  arsenophenylglycin. 

It  is  to  be  remembered  that,  during  or  immediately  after  active 
treatment  with  salvarsan  or  mercury,  the  Wassermann  reaction  may  be 
negative,  even  though  the  patient  is  not  cured.  As  a  general  rule,  a 
negative  reaction  under  these  conditions  should  not  be  considered  of 
value  unless  all  treatment  has  been  omitted  for  at  least  two  weeks; 
even  then  the  test,  if  negative,  should  be  repeated  a  month  or  so  later. 


MODIFICATIONS    OF    THE    WASSERMANN    REACTION  495 

Craig  has  recently  drawn  attention  to  the  fact  that  in  frank  untreated 
cases  the  degree  of  the  reaction  may  vary  within  wide  limits,  and  this  is 
especially  true  if  the  patients  are  receiving  active  treatment. 

Provocatory  Stimulation. — Paradoxic  as  it  would  at  first  appear, 
antisyphilitic  treatment  may  convert  a  negatively  reacting  serum  into 
a  positive  one.  In  not  a  few  cases  of  latent  syphilis  reacting  negatively 
the  administration  of  a  specific  spirillicidal  agent,  such  as  mercury  or 
salvarsan,  is  followed  by  positive  reactions,  due  probably  to  the  libera- 
tion of  endotoxins  from  destroyed  spirochetes  or  to  a  stimulation  of  the 
spirochetes  by  a  dose  of  drug  that  did  not  suffice  to  kill  them.  This 
condition  is  analogous  to  the  Herxheimer  reaction,  or  the  aggravation 
of  skin  lesions  sometimes  observed  to  follow  the  administration  of 
mercury  or  salvarsan.  The  fact  possesses  practical  value,  for  in  cases 
where  lues  is  known  to  have  been  present  or  is  strongly  suspected,  and 
the  Wassermann  reaction  is  indefinite  or  negative,  the  administration  of 
0.3  to  0.4  gram  of  salvarsan  or  neosalvarsan,  followed  by  a  Wassermann 
reaction  three  days  later,  may  now  show  a  positive  reaction  and  thus 
indicate  a  latent  syphilis  requiring  further  treatment. 

PRACTICAL  VALUE  OF  THE  WASSERMANN  REACTION 

As  previously  stated,  the  Wassermann  reaction  serves  two  important 
purposes:  (1)  As  an  invaluable  aid  in  the  diagnosis  and  (2)  as  a  guide 
in  the  treatment  of  syphilis. 

The  reaction  may  be  of  great  value  in  determining  the  diagnosis  of 
extragenital  sores  and  of  atypical  lesions  in  all  stages  of  syphilis.  A 
negative  reaction,  however,  has  less  value  than  a  positive  one,  and 
whenever  possible,  a  microscopic  examination  of  the  secretions  with  the 
dark-field  illuminator  should  be  made  in  order  to  confirm  the  diagnosis. 
In  early  latent  syphilis,  after  the  initial  lesion  has  healed,  and  before 
the  secondary  eruption  appears,  the  Wassermann  reaction  is  frequently 
the  only  means  of  making  the  diagnosis,  especially  if  the  chancre  has 
been  small,  atypical,  and  practically  neglected. 

Indefinite  symptoms  and  clinical  unrecognizable  cases  constitute  a 
considerable  proportion  of  cases  of  syphilis,  and,  as  is  true  in  all  other 
infections,  this  class  constitutes  the  greatest  menace  to  public  health. 
Many  patients  are  sincere  in  denying  knowledge  of  infection  and  early 
symptoms  may  be  overlooked,  the  Wassermann  reaction  being  the  sole 
means  of  diagnosis  and  serving  in  this  connection  as  an  invaluable  aid. 

Usually  the  symptoms  of  syphilis  are  so  well  marked  in  the  secondary 
stage  that  the  reaction  is  in  most  instances  but  confirmatory  evidence. 


496  THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

However,  in  doubtful  cases  a  negative  reaction  excludes  syphilis  with 
almost  absolute  certainty,  especially  if  the  reaction  is  repeatedly 
negative. 

In  the  late  latent  and  tertiary  stages  of  syphilis  the  Wassermann 
reaction  may  be  the  only  available  basis  on  which  to  establish  a  diagnosis. 
When  one  remembers  how  varied  are  the  clinical  manifestations  of 
chronic  syphilis,  how  wide-spread  is  the  disease,  and  how  frequently  the 
reaction  establishes  the  true  diagnosis,  the  reaction  must  be  regarded  as 
being  of  great  value  and  as  an  indispensable  diagnostic  aid.  It  must  not 
be  forgotten  that  patients  showing  an  early  involvement  of  the  central 
nervous  system,  and  even  those  showing  no  such  symptoms,  may  react 
negatively  with  blood-serum  and  positively  with  spinal  fluid;  in  all 
such  cases  the  spinal  fluid  should  be  examined  whenever  possible. 

A  positive  reaction  occurring  in  aborting  women  is  an  indication  for 
treatment  and  may  protect  the  fetus.  Similarly  a  positive  reaction  in 
either  parent  of  a  seemingly  healthy  infant  is  an  indication  for  treatment 
of  the  child  especially  if  the  mother  reacts  positively. 

In  this  connection,  however,  one  point  is  worthy  of  special  emphasis, 
namely,  that  although  a  positive  reaction  indicates  that  the  patient  is 
luetic,  it  does  not  necessarily  mean  that  a  particular  lesion  is  syphilitic. 
For  example,  a  person  may  be  luetic  and  yet  have  a  cancerous  ulceration 
of  the  larynx.  The  mere  fact  that  the  lesion  does  not  improve  under 
antisyphilitic  treatment  does  not  detract  from  the  value  of  the  Wasser- 
mann reaction,  but  is  a  warning  that  more  care  is  required  in  making  the 
clinical  examination.  I  have  seen  a  number  of  such  cases  in  which  a 
positive  Wassermann  reaction  was  held  a  priori  as  evidence  of  the  syphil- 
itic nature  of  a  lesion  that  later  proved  to  be  either  malignant  or  tuber- 
culous. A  weak  positive  reaction,  associated  with  an  active  ulcerating 
lesion,  very  frequently  indicates  that  the  lesion  is  not  syphilitic,  for 
active  lesions  usually  yield  strongly  positive  reactions. 

In  this  connection  may  also  be  mentioned  the  growing  importance 
the  Wassermann  reaction  has  assumed  in  life-insurance  examinations. 
Statistics  show  that  from  one-tenth  to  one-third  of  all  persons  infected 
with  syphilis  die  as  the  results  of  the  disease,  and  the  death-rate  among 
5000  syphilitics  accepted  for  insurance  was  one-third  over  expectation 
(Brockbank). 

An  important  question,  especially  from  the  standpoint  of  thera- 
peutics is :  Does  a  positive  reaction  invariably  indicate  the  presence  of 
living  spirochetes?  May  the  reaction  remain  positive  for  an  indefinite 
time  after  the  patient  has  been  cured,  just  as  agglutinins  and  antitoxins 


MODIFICATIONS    OF   THE    WASSERMANN    REACTION  497 

may  persist  in  the  blood  for  some  time  after  recovery  from  typhoid  fever 
and  diphtheria  has  taken  place?  The  sum  total  of  the  experience  of 
investigators  from  all  parts  of  the  world  would  indicate  that  a  persist- 
ently positive  reaction  means  the  presence  of  living  spirochetes  some- 
where in  the  body.  The  lesions  may  not  be  active;  the  patient,  while 
clinically  healthy,  may  be  infective,  and  is  always  subject  to  possible 
recurrences  of  clinical  syphilis. 

Although  gummas  are  slightly  infectious,  it  is  now  known  that  they 
contain  living  spirochetes,  and  the  former  view,  which  regarded  them  as 
sequels,  rather  than  as  actual  active  lesions  of  syphilis,  is  no  longer 
tenable. 

Just  how  long  the  reaction  may  remain  positive  after  the  patient  is 
actually  cured  and  all  spirochetes  are  dead  is,  of  course,  difficult  to  state, 
but  experimental  studies  on  the  lower  animals  has  shown  that  the  reagin 
disappears  somewhat  quickly  under  these  conditions. 

Although  a  persistently  negative  reaction  is  of  good  prognostic 
importance,  it  is  not  so  conclusive  in  the  information  it  yields  as  is  a 
positive  reaction.  In  other  words,  an  occasional  active  lues  may  react 
negatively,  and  not  infrequently  active  syphilitic  lesions  are  found  at 
autopsy  in  persons  whose  blood  reacted  negatively  during  life.  While 
it  is  true  that  great  harm  may  result  from  a  false  positive  diagnosis  due 
to  faulty  technic,  yet  it  must  be  admitted  that  the  Wassermann  reac- 
tion is  not  too  delicate,  and  that  we  are  just  as  prone  to  err  on  the  side 
of  securing  too  many  negative  reactions.  Every  effort  should  be  made 
to  render  the  test  as  delicate  as  is  possible  with  specificity. 

While  the  value  and  dependability  of  the  Wassermann  reaction  are 
based  upon  skilful  technic  that  will  eventually  limit  the  performance 
of  the  test  to  specially  trained  persons  in  central  laboratories,  every 
effort  should  be  made  to  render  accessible  to  all  persons  this  valuable 
diagnostic  test  of  a  disease  that  has  such  great  social  and  economic 
importance.  At  present  many  persons  are  unable  to  afford  the  expense 
of  a  number  of  tests,  or  even  of  one  test,  as  required  in  the  modern 
treatment  of  this  disease.  This  deficiency  should  be  corrected,  and  the 
test  made  available  in  all  free  dispensaries,  especially  those  under  the 
supervision  of  a  Social  Service  Department. 
32 


CHAPTER  XXIV 

THE  TECHNIC  OF  COMPLEMENT-FIXATION  REACTIONS 

(Continued) 

Specific  Complement  Fixation  in  Bacterial  Diseases. — As  has  been 
stated  elsewhere,  the  first  complement-fixation  tests  were  performed  by 
Bordet  with  bacterial  antigens  and  antiserums  (pest  and  typhoid). 
Following  the  application  of  the  principles  of  complement  fixation  in  the 
serum  diagnosis  of  syphilis,  it  was  but  natural  that  the  possibilities  of 
this  method  as  a  general  means  of  diagnosis  soon  became  appreciated, 
and  in  a  short  time  numerous  infections  were  studied. 

Probably  in  no  disease  has  complement  fixation  proved  so  constant 
or  so  valuable  a  diagnostic  procedure  as  in  syphilis.  In  this  condition 
the  peculiar  lipodophilic  reagin  is  largely  responsible  for  the  marked 
fixation  of  complement,  and  from  our  present  knowledge  on  the  subject 
we  learn  that  this  phenomenon  has  practically  no  analogy  in  any  other 
disease  except  frambesia. 

With  few  exceptions  bacterial  antigens  are  likely  to  yield  weaker  and 
more  inconstant  reactions.  This  is  due  to  the  fact  either  that  our 
antigen  lacks  a  more  available  and  specific  antigenic  principle,  or  that 
the  amount  of  complement-fixing  bodies  is  small  and  variable.  For 
these  reasons  it  becomes  apparent  that  the  preparation  of  antigen  and 
delicacy  of  technic  are  highly  important  factors. 

Preparation  of  Bacterial  Antigens. — Either  the  endotoxins  or  whole 
bacterial  body  may  constitute  the  main  portion  of  an  antigen.  Most 
recent  efforts  have  aimed  thoroughly  to  disorganize  the  bacterial  cell  in 
order  to  liberate  the  endotoxic  substances  that  pass  into  solution  and 
constitute  the  antigen.  Experience  has  frequently  shown,  however, 
that  the  protein  substances  of  the  bacterial  cell  itself  possess  antigenic 
properties,  and  accordingly  I  have  generally  found  that  antigens  com- 
posed of  cells  and  the  products  of  cellular  activity  are  usually  more 
satisfactory  than  those  prepared  of  the  endotoxic  substances  alone. 

As  a  general  rule,  bacterial  antigens  should  be  polyvalent — i.  6.,  made 
up  of  a  number  of  different  strains  of  the  same  microorganism.  Recent 
researches  in  bacteriology  tend  to  show  that  different  strains  of  the 

498 


PREPARATION    OF    BACTERIAL   ANTIGENS  499 

same  microorganism  have  particular  and  more  or  less  individual  patho- 
genic and  sometimes  biologic  characteristics,  and  it  is  reasonable  to 
assume  that  the  antibody  will  likewise  show  individual  properties  and  a 
special  affinity  for  its  particular  antigen.  When,  therefore,  one  antigen 
is  being  used  in  complement-fixation  work,  the  results  are  more  likely 
to  be  satisfactory  if  a  large  number  of  different  strains  are  included  in 
the  antigen,  with  the  hope  that  at  least  one  of  them  will  show  a  par- 
ticular affinity  for  the  antibody  in  the  patient's  serum. 

Bacterial  antigens  may  be  prepared  in  various  ways. 

First  Method. — Cultures  are  grown  in  a  suitable  fluid  medium,  such 
as  plain  bouillon,  for  forty-eight  hours,  or  upon  a  solid  medium,  and 
washed  off  with  a  suitable  quantity  of  normal  saline  solution.  The 
culture  or  emulsion  is  shaken  for  an  hour  or  so  to  break  up  the  clumps, 
and  then  heated  to  60°  C.  for  an  hour.  It  is  preserved  by  the  addition 
of  1  per  cent  of  glycerin  and  0.5  per  cent,  of  phenol. 

This  constitutes  the  simplest  bacterial  antigen.  It  is  composed  of 
both  bacterial  cells  and  the  products  of  bacterial  activity,  and  frequently 
yields  uniform  and  satisfactory  results. 

Second  Method. — Cultures  are  grown  on  a  suitable  solid  medium  for 
from  twenty-four  to  forty-eight  hours.  Growths  are  removed  by  adding 
sufficient  distilled  water  or  normal  saline  solution  to  yield  a  milky  sus- 
pension. The  emulsion  is  heated  to  56°  C.  in  a  water-bath  for  one  hour, 
followed  by  one  hour  at  80°  C.,  and  shaken  mechanically  with  glass 
beads  for  twenty-four  hours,  to  facilitate  disintegration.  It  is  then 
filtered  through  paper  pulp  and  a  sterile  Berkefeld  filter  with  a  neutral 
reaction,  or  thoroughly  centrifugalized;  the  filtrate  is  then  heated  at 
56°  C.  for  one-half  hour  on  each  of  three  successive  days  to  sterilize,  and 
preserved  with  0.5  per  cent,  phenol  and  used  as  antigen  after  being  di- 
luted and  made  isotonic  with  1  part  of  9  per  cent,  salt  solution  to  9 
parts  of  the  antigen. 

This  antigen  is  composed  essentially  of  endotoxic  substances, 
and  is  the  one  usually  employed  in  the  preparation  of  gonococcus 
antigen. 

Third  Method. — Cultures  are  grown  on  a  solid  medium,  washed  off 
with  normal  saline  solution,  and  the  emulsion  centrifuged  thoroughly. 
The  sediment  is  dried  over  sulphuric  acid  or  calcium  chlorid,  and  the 
dried  material  thoroughly  ground  with  crystals  of  sodium  chlorid.  Suf- 
ficient distilled  water  is  then  added  to  render  the  solution  isotonic,  and 
so  that  it  will  contain  about  0.05  gram  of  dried  material  in  each  cubic 


500  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

centimeter.  This  emulsion  is  then  shaken  for  twenty-four  nours, 
filtered  or  centrifuged,  the  filtrate  preserved  with  0.5  per  cent,  of  phenol, 
and  used  as  antigen. 

Fourth  Method. — Cultures  are  grown  on  a  solid  medium  and  washed 
off  with  normal  saline  solution.  Saline  suspension  is  then  precipitated 
with  an  equal  quantity  of  absolute  alcohol  and  centrifugalized.  The 
sediment  is  dried  in  vacuo  over  sulphuric  acid,  weighed,  and  ground  into 
a  fine  powder  with  sufficient  crystals  of  sodium  chlorid  to  make  a  2  per 
cent  suspension  of  dried  material  in  isotonic  saline  solution.  This 
stock  suspension  is  not  filtered  or  centrifuged,  but  is  further  diluted 
with  saline  solution,  and  constitutes  the  antigen  (method  of  Besredka, 
modified  by  Gay).  The  actual  amounts  of  dry  antigenic  substance 
contained  in  1  c.c.  of  various  dilutions  are  as  follows: 

1  c.c.  of  1:  40  dilution  =  0.5  mg. 

1  c.c.  of  1:  80  dilution  =  0.25  mg. 

1  c.c.  of  1:  160  dilution  =  0.125  mg. 

1  c.c.  of  1 :  320  dilution  =  0.062  mg. 

1  c.c.  of  1:  640  dilution  =  0.031  mg. 

1  c.c.  of  1:  1280  dilution  =  1.0155  mg.,  etc. 

Standardizing  Bacterial  Antigens. — After  an  antigen  has  been  pre- 
pared it  is  standardized  by  determining  the  anticomplementary  dose — 
i.e.,  the  amount  of  antigen  that  just  begins  to  show  inhibition  of  hemol- 
ysis  due  to  non-specific  complement  fixation.  This  dose  is  easily 
determined  by  adding  increasing  amounts  of  antigen  to  a  series  of  test- 
tubes  with  a  constant  dose  of  complement  in  each.  As  a  general  rule, 
it  is  well  to  add  to  each  tube  a  constant  dose  of  fresh  normal  inactivated 
serum,  e.  g.}  as  0.1  to  0.2  c.c.,  when  the  anticomplementary  action  of 
serum  alone  is  allowed  for.  I  would  emphasize  the  necessity  of  doing 
this  in  experimental  work  with  rabbit,  dog,  or  any  other  animal  serum. 
After  incubating  for  one  hour,  two  units  of  hemolytic  amboceptor 
and  1  c.c.  of  corpuscle  suspension  are  added  to  each  tube,  and  the 
tubes  are  reincubated  for  an  hour  or  two  and  the  reading  made.  In 
the  main  test,  one-quarter  to  one-half  the  anticomplementary  unit  may 
be  used,  as  this  amount  is  known  to  be  free  from  any  power  of  non- 
specific complement  fixation.  The  former  dose  is,  of  course,  safer  than 
the  latter. 

The  standardization  may  be  completed  by  determining  the  antigenic 
dose  of  the  antigen  by  titrating  with  a  suitable  and  constant  dose  of 
specific  immune  serum.  This  titration  is  conducted  by  placing  in  a 
series  of  test-tubes  increasing  doses  of  antigen  with  a  constant  dose  of 
heated  immune  serum  (usually  0.1  c.c.)  and  a  constant  dose  of  comple- 


PRINCIPLES  OF  COMPLEMENT  FIXATION  WITH  BACTERIAL  ANTIGENS     501 

ment.  After  an  hour  the  proper  dose  of  hemolytic  amboceptor  and  cor- 
puscles is  added.  The  readings  may  be  made  an  hour  or  two  later,  or 
after  the  tubes  have  been  allowed  to  settle  in  a  refrigerator.  That  tube 
showing  just  complete  inhibition  of  hemolysis  contains  the  antigenic  unit. 
For  the  main  test  it  is  well  to  use  double  this  amount,  providing  this 
dose  is  not  more,  and  preferably  less,  than  half  the  anticomplementary 
unit. 

The  antigenic  titration  is  not  always  satisfactory,  for  when  an  arti- 
ficial immune  serum  is  used  the  concentration  of  antibodies  may  yield 
a  much  stronger  reaction  than  one  would  expect  in  testing  human  serums. 
Further  than  this,  the  antibody  content  in  antiserums  varies  considerably 
so  that  the  antigenic  unit  fluctuates  according  to  the  particular  serum 
used  in  making  the  titration.  In  general,  therefore,  it  is  sufficient  to 
determine  the  anticomplementary  unit  and  to  use  half  or  quarter  this 
amount  in  performing  the  main  test. 

After  antigens  are  prepared  they  may  require  further  dilution  with 
saline  solution.  This  can  be  determined  only  by  experience  and  as  the 
result  of  a  trial  titration. 

As  watery  extracts  are  prone  to  deteriorate,  it  should  be  made  a  rule 
that  the  anticomplementary  dose  be  determined  each  time  before  the  main 
test  is  conducted. 

It  has  quite  generally  been  proved  that  alcoholic  extracts  of  bacteria 
do  not  yield  satisfactory  antigens,  despite  the  advantage  to  be  gained 
because  of  their  stability. 

Principles  of  Complement  Fixation  with  Bacterial  Antigens. — The 
principles  of  complement  fixation  in  general  should  be  thoroughly  under- 
stood. 

After  making  considerable  comparative  studies  with  various  methods, 
I  am  convinced  that,  in  the  final  analysis,  a  simple  technic  is  best.  I 
titrate  the  complement  or  use  a  relatively  small  but  safe  dose  of  com- 
plement,— 1  c.c.  of  a  1  :  20  dilution  ( =  0.05  c.c.  undiluted  serum), — and 
titrate  the  hemolytic  amboceptor  with  this  constant  dose  or  unit  of 
complement  and  the  corpuscle  suspension.  This  titration  is  made  each 
time  the  reactions  are  performed,  and  with  each  and  every  complement 
serum  and  corpuscle  suspension.  In  this  way  differences  in  the  activity 
of  different  guinea-pig  serums  are  readily  detected  and  adjusted.  If 
exactly  one  unit  of  complement  or  amboceptor  is  used  in  conducting  the 
main  test,  the  controls  are  not  likely  to  be  completely  hemolyzed  unless 


502  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

the  serum  contains  natural  antisheep  amboceptor,  for  the  serum  alone 
will  probably  be  slightly  anticomplementary.  For  this  reason  I  am 
accustomed  to  use  2-unit  doses  of  complement  or  amboceptor,  and  I 
secure  reactions  that  are  very  delicate  and  yet  sharp  and  clear  cut.  If 
one  wishes  to  use  exactly  one  unit  of  complement  and  one  unit  of  am- 
boceptor, the  serum  and  antigen  alone  should  be  controlled,  as  in  the 
fourth  method  of  performing  the  Wassermann  reaction  (p.  470).  I  fre- 
quently use  this  technic  in  the  gonococcus-fixation  test,  but,  as  a  rule, 
I  have  found  the  simpler  technic  herein  given  equally  sensitive  and 
reliable. 

In  this  chapter  are  considered  the  main  bacterial  infections  in  which 
complement  fixation  has  been  shown  to  possess  value  as  a  means  of 
diagnosis.  Complement  fixation  has  also  proved  of  value  in  the  diag- 
nosis of  animal  parasitic  diseases,  such  as  echinococcus  infection,  and 
in  the  differentiation  of  the  proteins.  With  each  of  these  the  special 
methods  for  preparing  the  antigen,  titrating  the  antigen,  and  conduct- 
ing the  test  are  given. 

Complement  Fixation  in  the  Differentiation  of  Microparasites. — 
Several  investigators  have  applied  the  technic  of  complement-fixation 
in  a  study  of  the  relationship  and  differentiation  of  closely  related  bac- 
teria, spirochetes,  trypanosomes,  and  other  microparasites.  These 
studies  have  usually  been  made  by  immunizing  rabbits  with  a  particular 
strain  and  using  the  immune  serum  in  complement-fixation  tests  with 
antigens  of  the  other  microparasites  under  study.  As  a  general  rule  com- 
plement-fixation is  most  marked  with  homologous  antigen  and  antiserum; 
relationship  is  studied  according  to  whether  or  not  complement-fixation 
occurs  with  the  other  antigens.  Besredka,1  Foix  and  Mallein,2  Swift 
and  Thro3  have  reported  that  immune  amboceptors  specific  for  different 
strains  of  streptococci  can  be  demonstrated  by  means  of  the  complement- 
fixation  test.  In  my  own  work  with  five  strains  of  streptococci,4  with 
the  specific  purpose  of  studying  the  relationship  of  the  streptococcus 
commonly  found  associated  with  scarlet  fever  to  the  group  of  strep- 
tococci, I  found  that  differentiation  among  these  was  possible  when 
using  high  dilution  of  the  immune  sera,  but  in  lower  dilutions  differen- 
tiation was  not  found  by  means  of  complement-fixation  tests,  the  re- 

1  Am.  de  Flnst.  Pasteur,  1904,  28,  363. 

2  Presse  Medicale,  1907,  15,  777. 

3  Arch.  Int.  Med.,  1911,7,  24. 

4  Arch.  Int.  Med.,  1912,  9,  220. 


COMPLEMENT    FIXATION    IN    GONOCOCCUS    INFECTIONS  503 

suits  indicating  either  that  this  scarlet  fever  streptococcus  belonged 
to  the  common  group  of  streptococci  or  that  the  complement-fixation 
test  was  a  group  reaction.  In  a  similar  study  of  the  diphtheria  group 
of  bacilli  I1  found  that  .the  pseudodiphtheria  bacillus  could  not  be  differ- 
entiated from  other  members  of  the  group  by  complement-fixation 
reactions  indicating  its  relationship  to  the  true  diphtheria  bacillus; 
similar  studies  made  by  Williams,  Raiziss,  and  I2  with  the  typhoid 
colon  group  of  bacilli,  by  Craig  and  Nickols3  with  several  strains  of 
spirochetes,  by  Cooke4  with  acid-fast  bacilli,  by  Olmstead5  and  Woll- 
stein6  with  meningococci,  by  Hooker7  with  typhoid  bacilli,  and  by 
Kitchens  and  Brown8  with  streptococci  indicate  that  the  bacterio- 
lytic  amboceptors  are  highly  specific  and  yield  highly  specific  reac- 
tions with  proper  technic,  and  particularly  with  good  and  sensitive 
antigens,  and  under  these  conditions  the  complement-fixation  test 
may  be  more  delicate  in  differentiation  than  agglutination  or  other 
immunity  reactions.  As  a  practical  procedure,  however,  the  comple- 
ment-fixation technic  is  too  intricate  and  time-consuming.  In  this  con- 
nection I  desire  to  direct  particular  attention  to  the  possibility  of  the  sera 
of  normal  rabbits  yielding  positive  complement-fixation  reactions  with 
various  bacterial  and  lipoidal  extracts  as  discussed  on  page  J+19,  in  con- 
ducting complement-fixation  tests  with  rabbit  or  dog  sera  the  serum  of  each 
animal  should  first  be  tested  with  the  antigen  and  immunization  conducted 
only  in  case  the  animal's  serum  is  found  to  react  negatively;  subsequent 
complement-fixing  properties  of  the  serum  may  then  be  ascribed  to  the  pres- 
ence of  specific  amboceptors. 


COMPLEMENT  FIXATION  IN  GONOCOCCUS  INFECTIONS 

This  was  one  of  the  first  infections  to  be  studied  by  means  of 
the  complement-fixation  technic,  but  the  results  secured  were  not 
generally  satisfactory  until  it  was  shown  that  the  antigen  must  be 
polyvalent. 

1  Jour.  Infect.  Dis.,  1912,  11,  44. 

2  Jour.  Infect.  Dis.,  1913,  13,  321. 

3  Jour.  Exper.  Med.,  1912,  16,  336. 

4  Jour.  Amer.  Med.  Assoc.,  1917,  Ixviii,  1504. 
6  Jour.  Immunology,  1916,  1,  307. 

6  Jour.  Exper.  Med.,  1914,  20,  201. 

7  Jour.  Immunology,  1916,  1,  1. 

8  Personal  communication. 


504  THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

Historic — In  1906  Muller  and  Oppenheim1  applied  the  complement-fixation  test 
to  the  diagnosis  of  gonorrheal  arthritis,  using  a  culture  of  the  gonococcus  as  antigen. 
To  these  observers,  therefore,  belongs  the  credit  of  being  the  first  to  record  a  comple- 
ment-fixation test  in  a  gonococcus  infection.  A  little  later  in  the  same  year  Carl 
Bruch2  applied  the  reaction  to  three  cases  of  gonorrhea,  using  the  serum  of  immunized 
rabbits,  and  reported  favorable  results.  In  1907  Meakins3  reported  having  secured 
positive  reactions  in  three  cases  of  gonorrheal  arthritis,  which  was  the  first  report  in 
America  published  on  this  subject.  Th.  Vanned4  studied  the  specificity  of  the  reac- 
tion with  the  serums  of  rabbits  immunized  with  gonococcus  protein  and  one  of  a 
meningococcus,  and  reported  that  the  meningococcus  immune  serum  did  not  show 
complement  fixation  with  gonococcus  antigen,  and,  vice  versa,  that  gonococcus  ambo- 
ceptor  was  not  bound  by  meningococcus  antigen.  Wollstein5  (1907),  in  a  study  of 
the  biological  relationship  of  the  gonococcus  and  meningococcus,  reported  findings 
differing  from  those  of  Vannod.  The  former  observer  found  that  bacteriolytic  am- 
boceptors  in  the  serums  of  rabbits  immunized  with  these  cultures  were  closely  related 
and  yielded  fixation  of  complement  with  either  antigen.  Teaque  and  Torrey,6  in 
1907,  issued  a  very  important  communication  showing  that  the  differences  in  results 
of  previous  investigators  were  probably  due  in  part  to  the  use  of  single  strains  of  the 
organisms  in  the  preparation  of  antigens  and  immune  serums.  They  emphasized 
the  fact  that  the  gonococcus  belongs  to  a  heterogeneous  family,  and  that  in  attempt- 
ing to  formulate  a  diagnosis  of  gonorrheal  infection  by  the  complement-fixation 
method,  the  extracts  of  several  different  strains  should  be  used.  Naz  Vannod  and 
later  Watabiki7  found  that  the  gonococcus  and  meningococcus  antibodies  were  quite 
specific  for  their  homologous  antigens  in  complement-fixation  reactions. 

Particular  attention  was  drawn  to  the  gonococcus  complement-fixation  test  by 
the  work  of  Schwartz  and  McNeal.8  These  investigators  emphasized  the  necessity 
of  using  polyvalent  antigens,  and  their  encouraging  reports  have  stimulated  renewed 
interest  in  this  subject.  They  found  that  if  the  infection  is  confined  to  the  anterior 
urethra,  a  positive  reaction  is  not  obtained;  that  a  strong  reaction  is  not  to  be  ex- 
pected before  the  fourth  week  of  the  infection,  and  then  only  in  acute  cases  with  com- 
plications. They  regard  a  positive  reaction  as  indicating  the  presence  or  recent 
activity  in  the  body  of  a  focus  of  living  gonococci,  although  a  negative  reaction  does 
not  exclude  gonococcus  infection.  The  test,  therefore,  has  a  more  positive  than  a 
negative  value.  With  Flexner's  antimeningococcus  serum  positive  reactions  re- 
sulted with  their  gonococcus  antigen;  with  serums  from  cases  of  cerebrospinal  menin- 
gitis (meningococcic)  the  results  were  negative. 

In  the  succeeding  years  numerous  investigators,  including  Swinburne,  Gradwohl, 
O'Neil,  Gardner  and  Clowes,  Thomas  and  Ivy,  Kolmer  and  Brown,  have  reported 
favorably  upon  the  practical  value  of  the  gonococcus  complement-fixation  test, 
particularly  as  an  aid  in  determining  whether  or  not  a  patient  is  cured  of  the 
infection. 

Technic. — Since,  because  of  the  comparatively  slight  cellular  in- 
volvement, the  quantity  of  antibody  produced  in  a  localized  gonococcus 
infection  is  probably  small,  the  complement-fixation  reactions  are 
generally  weak,  and  consequently  require  the  closest  technical  attention, 
especially  as  regards  the  preparation  of  antigen  and  accurate  adjust- 
ment of  the  hemolytic  system. 

Hemolytic  System. — As  a  rule,  the  antisheep  hemolytic  system  is 
employed;  the  various  ingredients  may  be  used  in  one-half  the  quantity 
employed  in  the  original  Wassermann  reaction,  as  given  in  the  preceding 

1  Wien.  klin.  Wochenschr.,  1906,  19,  894. 

2  Deutsch.  med.  Wochenschr.,  1906,  70,  36. 

3  Johns  Hopkins  Medical  Bull.,  1907,  18,  255. 

4  Zeitschr.  f.  Bakter.,  1907,  44,  10.  5  Jour.  Exp.  Med.,  1907,  9,  588. 

6  Jour.  Med.  Research,  1907,  17,  223.  7  Jour.  Infect.  Diseases,  1910,  7,  159. 

8Amer.  Jour.  Med.  Sci.,  May,  1911;  ibid.,  September,  1912;  ibid.,  December, 
1912. 


COMPLEMENT    FIXATION    IN    GONOCOCCUS    INFECTIONS  505 

chapter,  with  the  technic  ef  the  syphilis  reaction,  or  one-tenth  the  quan- 
tity employed  in  the  original  Wassermann  technic  may  be  employed. 
I  prefer  to  employ  the  larger  amounts  because  the  readings  are  usually 
easier  to  interpret. 

Fresh  guinea-pig  complement  serum  is  diluted  1  :  20  and  used  in  dose 
of  1  c.c.  (  =  0.05  c.c.  serum);  sheep's  corpuscles  are  made  up  in  a  2}^  per 
cent,  suspension  and  used  in  dose  of  1  c.c.;  antisheep  amboceptor  is 
titrated  (see  p.  399)  and  used  in  an  amount  equal  to  2  hemolytic  doses 
in  conducting  the  antigen  titration  and  in  the  test  proper. 

Kolmer  and  Brown1  have  compared  the  practical  value  of  the  anti- 
sheep  and  antihuman  hemolytic  systems  in  the  examination  of  a  number 
of  serums.  When  the  latter  were  used,  some  of  the  reactions  were 
somewhat  stronger  and  yielded  slightly  better  results,  showing  the 
influence,  probably,  of  natural  antisheep  amboceptor  present  in  a  large 
proportion  of  human  serums. 

Antigen. — This  constitutes  the  most  important  ingredient  of  the 
test.  As  Teague  and  Torrey  and  Schwartz  and  McNeal  have  em- 
phasized, the  antigen  should  be  prepared  of  many  different  strains  of 
gonococci.  The  difficulty  of  isolating  this  organism  and  the  constant 
care  required  in  subculturing  and  keeping  a  large  number  of  strains  alive 
render  it  practically  impossible  for  many  persons  to  prepare  a  gonococcus 
antigen.  Therefore  until  simpler  methods  are  devised,  this  antigen  is 
best  prepared  in  large  central  laboratories,  where  the  cultures  are  handled 
and  preserved  by  specially  trained  persons. 

The  gonococci  are  well  grown  on  a  salt-free  veal  agar,  neutral  in 
reaction  to  phenolphthalein,  and  to  which  a  few  drops  of  sterile  hydrocele 
fluid  may  be  added.  After  culturing  for  from  twenty-four  to  forty- 
eight  hours  the  growths  are  washed  off  with  distilled  water,  and  the 
emulsion  is  heated  in  a  water-bath  for  two  hours  at  56°  C.  It  is  then 
heated  at  80°  C.  for  one  hour,  filtered  through  paper  pulp  or  centrifu- 
galized,  and  passed  through  a  Berkefeld  filter  which  is  reserved  for  this 
purpose  alone  and  is  neutral  in  reaction  (see  page  78) .  A  small  amount 
of  preservative,  as,  e.  g.,  0.1  c.c.  of  a  1  :  100  dilution  of  phenol  to  each 
cubic  centimeter  of  antigen,  may  be  added.  The  antigen  is  then  well 
preserved  in  small  amounts  in  ampules  that  are  sealed  and  heated  to 
56°  C.  for  half  an  hour  on  three  successive  days.  Just  before  being 
used  the  antigen  is  made  isotonic  by  adding  1  part  of  a  10  per  cent,  salt 
solution  to  9  parts  of  antigen.  I  preserve  the  antigen  in  ampules  con- 
taining 1  c.c.,  and  after  removing  the  antigen  from  the  ampule  to  a  large 
test-tube,  add  1  c.c.  of  10  per  cent,  salt  solution,  and  dilute  the  whole 
1  Jour.  Infect.  Dis.,  1914,  15,  6. 


506 


THE   TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 


1  :  10  with  the  addition  of  8  c.c.  of  normal  salt  solution,  after  which  the 
anticomplementary  titration  is  made. 

In  this  method  of  preparing  antigen  the  endotoxins  constitute  the 
main  antigenic  principle.  Kolmer  and.  Brown,  after  an  experimental 
study  of  the  various  antigens,  found  that  a  simple  suspension  of  gono- 
cocci  in  salt  solution  yielded  slightly  better  results.  The  various 
strains  are  grown  for  from  forty-eight  to  seventy-two  hours,  and  are 
then  washed  off  with  sterile  saline  solution,  observing  particular  care 
not  to  include  portions  of  the  culture-medium.  The  suspension  is 
then  shaken  to  break  up  clumps,  and  heated  to  56°  C.  for  an  hour.  A 
small  amount  of  preservative  is  now  added,  and  the  antigen  stored  in 
1  c.c.  ampules.  Before  using  it  is  diluted  1  :  10  or  1  :  20,  and  titrated 
for  the  anticomplementary  dose. 

Alcoholic  extracts  of  gonococci  have  very  little  practical  value,  as 
alcohol  is  not  satisfactory  for  extracting  the  antigenic  principles  of 
bacteria.  Warden1  has  reported  good  results  with  a  new  lipoid  antigen. 

The  anticomplementary  dose  of  the  antigen  should  be  determined, 
and  one-half  or  one-quarter  this  amount  should  be  used  in  conducting 
the  main  test.  An  antigenic  titration  may  also  be  conducted  with  an 
antigonococcus  serum,  to  determine  the  antigenic  value  of  the  antigen, 
but  in  practice  it  is  sufficient  to  use  one-half  the  anticomplementary 
dose.  This  titration  should  be  conducted  and  the  antigen  standardized 
before  the  main  tests  are  adjusted. 

In  the  following  table  the  results  of  an  anticomplementary  titration 
of  a  gonococcus  antigen  are  given,  the  approximate  dose  having  been 
ascertained  in  previous  titrations  (Fig.  120). 

TABLE  17.— ANTICOMPLEMENTARY  TITRATION  OF  A  GONOCOCCUS 

ANTIGEN 


<rfe 

SHEEP'S 

h 

COM- 

o^ 

ANTI- 

COR- 
PUS- 

<4-f 
Mri 

TUBE 

ANTIGEN, 
1:10,  C.c. 

PLE- 
MENT, 

(NO 
0*S 

SHEEP 
AMBO- 

CLES 
(2.5 

1 

RESULTS 

1:  20, 

-M,Q 

CEPTOR, 

PER 

_g 

C.c. 

JS  § 

UNITS 

CENT), 

Sd 

s.S 

C.c. 

"***d 

"rt  o 

1 

02 

1 

|s 

2 

1 

'gcc 

Complete  hemolysis 

2.  . 
3   , 

0.4 
0.6 

1 
1 

.sld 

o3  ° 

2 
2 

1 
1 

li 

Complete  hemolysis 
Complete  hemolysis 

4  

0.8 

1 

o^co 

2 

1 

I-3 

Slight    inhibition    of 

•.£  a,  -M 

CO  ^ 

hemolysis 

5  

1.0 

1 

"o  *  ^ 

2 

1 

1 

Marked  inhibition  of 

BD   CD   S3 

OJ^   0 

J" 

hemolysis 

6  

0 

1 

CH     ^  ""^ 

2 

1 

Hemolytic  control 

3"^ 

H 

Complete  hemolysis 

1  Jour.  Amer.  Med.  Assoc.,  1915,  Ixv,  2080;  Jour.  Lab.  and  Clin.  Med.,  1916,  i,  333: 


FIG.  120. — ANTICOMPLEMENTARY  TITRATION  OF  A  GONOCOCCTJS  ANTIGEN. 

The  tube  containing  0.6  c.c.  shows  slight  inhibition  of  hemolysis,  and  this  amount  is 

the  anticomplementary  unit. 


COMPLEMENT    FIXATION    IN    GONOCOCCUS    INFECTIONS  507 

If  the  antigen  is  new  and  the  anticomplementary  dose  is  entirely 
unknown,  it  may  be  necessary,  in  making  this  titration,  to  use  a  different 
dilution,  with  higher  and  lower  doses.  In  conducting  the  main  test  the 
foregoing  antigen  could  be  used  in  dose  of  0.2  or  0.4  c.c.  of  this  dilution. 

The  Test. — The  serums  should  be  fresh  and  clear,  and  heated  to  56°  C. 
for  one-half  hour.  For  each  serum  use  four  test-tubes  (12  by  1  cm.), 
arranged  in  a  row.  Into  each  of  the  first  three  place  the  dose  of  antigen 
and  increasing  doses  of  serum — 0.05  c.c.,  0.1  c.c.,  0.2  c.c.;  the  fourth 
tube  is  the  serum  control,  and  into  this  is  placed  the  maximum  dose  of 
serum  (0.2  c.c.)  but  no  antigen;  1  c.c.  of  complement  diluted  1  :  20  is 
added  to  each  tube.  The  following  controls  are  included: 

1.  A  positive  control  with  an  antigonococcus  serum  or  with  the  serum 
of  a  patient  who  reacted  positively  on  a  former  occasion. 

2.  A  negative  control  with  the  serum  of  a  healthy  person. 

Both  of  these  controls  may  be  set  up  with  but  the  maximum  dose  of 
serum  (0.2  c.c.). 

3.  The  serum  control  of  each  serum  is  conducted  in  the  fourth  tube  of 
each  series.     At  the  completion  of  the  test  this  tube  should  show  com- 
plete hemolysis  and  thereby  indicate  that  the  serum  was  not  anticom- 
plementary. 

4.  The  antigen  control  at  this  time  includes  the  dose  of  antigen  and 
complement. 

5.  The  hemolytic  system  control  at  this  time  receives  the  dose  of 
complement. 

6.  The  corpuscle  control  receives  1  c.c.  of  the  corpuscle  suspension. 
To  each  tube  sufficient  saline  solution  is  added  to  bring  the  total 

volume  up  to  about  2  c.c.  The  tubes  are  shaken  and  incubated  for  one 
hour  at  37°  C.  in  the  thermostat  or  in  a  water-bath  (not  less  than  one 
hour),  when  2  units  of  antisheep  amboceptor  and  1  c.c.  of  sheep  corpuscle 
suspension  are  added  to  each  tube  except  the  corpuscle  control.  The 
tubes  are  gently  shaken  again  and  reincubated  for  an  hour  or  longer, 
depending  upon  the  hemolysis  of  the  controls,  after  which  the  results 
are  recorded.  This  secondary  incubation  may  be  omitted  and  the  tubes 
placed  in  a  refrigerator  overnight  and  the  results  read  the  next  morning. 
Under  these  conditions  hemolysis  occurs  slowly,  and,  according  to  some 
workers  in  this  field,  the  reaction  becomes  more  delicate. 

Conducting  the  primary  incubation  by  placing  the  tubes  in  a  refrigerator 
at  8°  C.  for  four  hours  or  over  night  after  the  method  of  McNeal,  followed  by 
the  addition  of  2  units  of  hemolysin  and  1  c.c.  of  corpuscle  suspension  to 
each  tube  and  incubation  in  a  water-bath  at  38°  C.  for  one-half  to  one  hour, 


508 


THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 


yields  particularly  delicate  reactions  and  intensifies  the  degree  of  specific 
fixation  of  complement. 

The  following  table  is  an  example  of  a  gonococcus  fixation  test  with 
the  serum  of  a  case  of  gonorrheal  arthritis  (Fig.  121). 

TABLE  18.— GONOCOCCUS  COMPLEMENT-FIXATION  TEST 


SHEEP 

COR- 

COM- 

1 

ANTI- 

PUS- 

PATIENT'S SERUM, 

ANTIGEN, 

PLE- 

SHEEP 

CLES 

RESULTS   AFTER   ONE   AND  A 

C.c. 

1:10,  C.c. 

MENT, 

*O 

AMBO- 

(2.5 

HALF  HOURS  INCUBATION 

1:20, 

— 

CEPTOR, 

PER 

C.c. 

o3 

UNITS 

CENT), 

1 

C.c. 

0.05 

0.2 

1 

02 

2 

1 

Slight  inhibition  of  hem- 

j>»o 

olysis 

0.1 

0.2 

1 

+3  0 

2 

1 

Marked     inhibition     of 

faC^ 

hemolysis 

0.2 

0.2 

1 

49 

QJ   c3 

2 

1 

Complete    inhibition    of 

o3  SH 

hemolysis 

0.2 

0 

1 

Jo 

2 

1 

Serum  control:    hemoly- 

^ 

sis 

Positive    serum, 

0.2 

0.2 

1 

•-   *H 

2 

1 

Complete    inhibition    of 

««2 

hemolysis 

0.2 

0 

1 

<s^ 

2 

1 

Serum  control:    hemoly- 

« ~cS 

sis 

Negative  serum, 

m^ 

0.2 

0.2 

1 

fl 

2 

1 

Hemolysis 

0.2 

0 

1 

'•+3 

2 

1 

Hemolysis 

0 

0.2 

1 

'o 

2 

1 

Antigen    control  :     hem- 

§ 

olysis 

0 

0 

1 

0) 

2 

1 

Hemolytic           control: 

Js 

hemolysis 

0 

0 

0 

c» 

0 

1 

Corpuscle    control:     no 

hemolysis 

In  reading  the  results  the  controls  are  first  examined  and  should  show 
complete  hemolysis;  the  test  is  reported  as  negative  if  all  tubes  are 
hemolyzed,  weakly  positive  if  the  largest  dose  only  of  serum  (0.2  c.c.) 
shows  inhibition  of  hemolysis,  moderately  positive  if  the  0.1  and  0.2  c.c. 
doses  of  serum  show  inhibition  of  hemolysis,  and  strongly  positive  if 
the  0.05,  0.1,  and  0.2  c.c.  doses  of  serum  react  positively. 

The  test  may  be  conducted  with  but  one  dose  of  serum,  namely, 
0.2  c.c.  with  the  antigen  and  0.2  or  0.3  c.c.  in  the  serum  control  tube, 
after  the  manner  of  the  original  Wassermann  reaction;  in  this  case  the 
readings  are  made  after  the  usual  +,  +  +,  +  +  +,  and  +  +  +  + 
method  (see  page  464) . 

GONOCOCCUS  FIXATION  TEST,  USING  ONE-TENTH  THE  USUAL  AMOUNTS 

This  technic  is  employed  for  purposes  of  economy,  especially  since 
the  antigen  is  likely  to  be  expensive.  Otherwise  the  method  is  less 


FIG.  121.  —  GONOCOCCUS  COMPLEMENT-FIXATION  REACTION. 
Shows  a  +  reaction  with  0.05  c.c.  serum;  a  +  +  with  0.1  c.c.,  and  a 
0.2  c.c.,  a  strongly  positive  reaction. 


with 


COMPLEMENT   FIXATION   IN   GONOCOCCUS    INFECTIONS  509 

desirable  than  the  preceding  one,  as  the  results  are  more  difficult  to 
read. 

Complement  serum  is  diluted  1  :  10  and  used  in  dose  of  0.1  c.c.; 
corpuscles  are  made  up  in  a  10  per  cent,  suspension  and  used  in  dose  of 
0.1  c.c.,  the  amboceptor  is  titrated  with  these  amounts  of  complement 
and  corpuscles,  and  used  in  dose  equal  to  two  units.  Each  day,  before 
the  main  tests  are  undertaken,  the  anticomplementary  dose  of  antigen 
is  determined  by  placing  increasing  doses  of  diluted  antigen  with  com- 
plement and  salt  solution  in  a  series  of  tubes,  incubating  for  an  hour, 
adding  two  units  of  amboceptor  and  the  corpuscles,  followed  by  in- 
cubation for  another  hour.  One-half  or  one-quarter  of  the  anticom- 
plementary dose  is  used  in  making  the  main  test.  The  serums  are 
inactivated  and  used  in  three  ascending  doses, — 0.005,  0.01,  and  0.02  c.c., 
—equivalent  respectively  to  0.5,  1,  and  2  c.c.  of  a  1  :  100  dilution  (0.1  c.c. 
serum.  9.9  c.c.  salt  solution).  The  fourth  tube  of  each  series  contains 
the  maximum  dose  of  serum  without  antigen,  and  is  the  serum  control. 
The  other  controls,  general  technic,  and  readings  of  the  reaction  are 
the  same  as  those  previously  described. 

SPECIFICITY  OF  THE  GONOCOCCUS  COMPLEMENT-FIXATION  TEST 
Viewed  from  a  practical  standpoint,  the  reaction  is  highly  specific. 
While  complement-fixation  experiments  with  antigens  of  gonococci  and 
meningococci  and  their  respective  immune  serums  have  demonstrated  a 
biologic  relationship  between  these  microorganisms,  yet  practically  with 
human  serums  an  antigen  of  pure  cultures  of  gonococci  will  fix  com- 
plement only  with  the  gonococcus  antibody  (amboceptor).  In  this 
technic  a  specific  antigen  is  employed,  and  it  is,  therefore,  a  true  applica- 
tion of  the  Bordet-Gengou  reaction  of  complement  fixation  by  specific 
antigen  and  specific  antibody  (amboceptor).  Obtained  under  proper 
technical  conditions,  a  positive  reaction  is  invariably  reliable,  and  indi- 
cates the  presence  of  a  focus  of  living  gonococci. 

PRACTICAL  VALUE  OF  THE  GONOCOCCUS  COMPLEMENT-FIXATION  TEST 
From  our  present  knowledge  of  this  reaction,  it  may  be  stated : 

1.  That  the  difficulty  of  isolating  and  preserving  a  sufficient  number 
of  cultures  of  true  gonococci  in  order  to  prepare  a  satisfactory  poly- 
valent antigen  constitutes  a  weighty  drawback  to  the  practical  use  of 
the  test. 

2.  Because  the  gonococcus  antibody,  unless  complicated  by  wide- 
spread gonococcal  metastases,   is  produced  in  small  amount  in  the 


510  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

majority  of  cases,  the  degree  of  complement  fixation  is  usually  much  less 
than  that  which  occurs  in  the  syphilis  reaction,  and  accordingly  the 
reactions  are  usually  weaker  and  often  indefinite.  A  negative  reaction 
does  not  exclude  gonorrhea. 

3.  The  reaction  is  seldom  positive  during  the  first  four  to  six  weeks 
of  an  acute  anterior  or  posterior  urethritis,  in  the  absence  of  complica- 
tions.    In  acute  exacerbations  of  a  chronic  urethritis  the  reaction  is 
positive  in  about  80  per  cent,  of  cases.     In  ordinary  chronic  urethritis 
with  mild  infection  of  the  prostate  gland  the  reaction  is  positive  in  from 
30  to  40  per  cent,  of  cases.     In  chronic  urethritis  complicated  by  marked 
involvement  of  the  prostate  gland  and  epididymitis  the  reactions  are 
frequently  positive,  occurring  in  from  50  to  over  80  per  cent,  of  cases. 

The  test  possesses  considerable  value  in  determining  the  fitness  of  an 
applicant  for  a  marriage  license,  and  will,  no  doubt,  be  employed  for  this 
purpose  quite  extensively,  as  a  positive  reaction  is  now  generally  re- 
garded as  indicating  the  presence  of  a  focus  of  active  gonococcal 
infection. 

4.  During  the  course  of  an  acute  or  a  subacute  urethritis  the  occur- 
rence of  an  acute  complication,  such  as  prostatitis,  epididymitis,  etc., 
is  likely  to  result  in  a  positive  fixation  test. 

5.  In  gonorrheal  iritis  a  positive  reaction  occurs  in  over  80  per  cent, 
of  cases. 

6.  A  positive  reaction  may  persist  for  several  weeks  after  the  patient 
is  clinically  cured.    Torrey1  has  shown  experimentally  that  the  antibody 
persists  in  the  blood  of  rabbits  artifically  immunized  for  from  ten  days 
to  six  or  seven  weeks.    Usually,  under  proper  treatment,  the  reaction  in 
ordinary  cases  of  urethritis  disappears  in  from  two  to  three  weeks;  if, 
however,  a  positive  reaction  persists,  a  focus  of  infection  is  probably 
present,  and  the  patient  should  be  kept  under  further  observation  and 
the  treatment  persisted  in. 

7.  In  women  the  reaction  is  seldom  positive  until  the  infection  has 
reached  the  cervical  canal.    In  the  case  of  little  children,  however,  we 
have  known  positive  reactions  to  occur  in  acute  and  chronic  vulvo- 
vaginitis,  indicating  either  that  the  disease  is  more  severe  in  children, 
with  more  antibody  formation,  or  that  it  may  reach  the  cervical  canal. 

The  reaction  is  positive  in  about  60  per  cent,  of  cases  of  pyosal- 
pingitis,  and  the  test  may  prove  of  value  in  making  the  differentiation  of 
inflammatory  lesions  from  certain  cystic  and  neoplastic  conditions,  and 
in  establishing  the  gonorrheal  basis  of  many  of  these  infections. 
1  Jour.  Med.  Research,  1910,  i,  95. 


COMPLEMENT-FIXATION   TEST    IN    GLANDERS  511 

8.  Cases  of  gonorrheal  arthritis  yield  from  80  to  100  per  cent,  of 
positive  reactions,  and  the  'complement-fixation  test  has  considerable 
value  in  establishing  the  diagnosis  of  these  infections. 

9.  The  administration  of  gonococcus  vaccine  and  antigonococcus  serum 
is  likely  to  be  followed  by  positive  reactions.     Just  how  long  the  anti- 
bodies may  persist  in  the  blood  after  a  clinical  cure  has  been  effected 
it  is  difficult  to  state;  at  least  from  six  to  twelve  weeks'  time  should  be 
given  for  them  to  disappear. 

10.  In  medicolegal  cases  the  courts  may  not  accept  the  usual  evi- 
dence offered  by  a  bacteriologic  diagnosis  based  upon  stained  smears  of 
a  secretion,  and  cultures  are  frequently  differentiated  from  other  Gram- 
negative  diplococci  only  with  difficulty.     Conducted  with  the  proper 
technic,  the  gonococcus  fixation  test  is  highly  specific  and  much  less 
difficult  to  perform. 

11.  Finally,  it  must  be  emphasized  that  the  reaction  has  a  far  more 
positive  than  negative  value.     The  reaction  is  highly  specific,  but  there 
is  a  limit  to  its  delicacy,  so  that  a  negative  reaction  in  urethritis  does 
not  exclude  the  possibility  of  gonococcal  infection. 


COMPLEMENT-FIXATION  TEST  IN  GLANDERS 

The  complement-fixation  test  is  used  extensively  by  veterinarians 
in  making  a  laboratory  diagnosis  of  glanders.  The  test  has  been  found 
very  reliable,  and  is  usually  more  delicate  than  the  agglutination  test 
and  the  Strauss  guinea-pig  test.  It  has  also  been  used  successfully  in 
the  diagnosis  of  human  glanders. 

Preparation  and  Standardization  of  Antigen. — The  antigen  should  be 
polyvalent,  and  composed  of  at  least  several  different  strains.  Cultures 
of  Bacillus  mallei  are  grown  on  slants  of  glycerin  agar  (1.6  per  cent,  acid) 
for  from  forty-eight  to  seventy-two  hours.  The  growths  are  then 
removed,  and  sufficient  distilled  water  added  to  give  a  milky  suspension. 
This  suspension  is  sterilized  by  heating  the  tubes  to  60°  C.  for  two  hours. 
They  are  then  shaken  mechanically  with  glass  beads  for  a  few  hours  on 
two  successive  days.  Enough  sodium  chlorid  is  added  to  make  the 
solution  isotonic,  and  the  whole  is  preserved  with  0.5  per  cent,  phenol 
and  stored  in  a  dark,  cold  place,  where  it  will  keep  for  many  months. 
Antigen  may  also  be  prepared  in  the  same  manner  as  gonococcus  anti- 
gen as  described  on  page  505. 

A  simpler  antigen  is  prepared  by  growing  the  bacillus  in  glycerin 
bouillon  for  seventy-two  hours,  sterilizing  by  heating  to  60°  C.  for  two 
hours,  and  preserving  with  the  addition  of  0.5  per  cent,  of  phenol. 


512  THE    TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

The  anticomplementary  dose  is  then  determined  by  tit-ration.  The 
antigen  is  diluted  1  : 20  by  mixing  1  c.c.  with  19  c.c.  of  normal  saline 
solution.  To  a  series  of  seven  test-tubes  add  increasing  amounts  of 
diluted  antigen  as  follows:  0.1,  0.2,  0.4,  0.6,  0.8,  1,  and  2  c.c.  Add  1  c.c. 
of  complement  serum  (1  : 20)  to  each  tube,,  and  sufficient  salt  solution 
to  bring  the  total  volume  in  each  up  to  3  c.c.  Incubate  for  an  hour  at 
37°  C.,  and  add  2  units  of  antisheep  amboceptor  titrated  just  previous 
to  making  the  test  (see  p.  399)  and  1  c.c.  of  sheep  corpuscle  suspension. 
Reincubate  for  an  hour  or  an  hour  and  a  half.  At  the  end  of  this  time 
that  tube  showing  beginning  inhibition  of  hemolysis  contains  the  anticom- 
plementary unit,  and  one-fourth  to  one-half  this  amount  is  used  in  making 
the  main  test. 

An  antigenic  titration  may  also  be  made,  but  this  is  not  absolutely 
necessary.  To  a  series  of  tubes  containing  0.05,  0.1,  0.15,  0.2,  0.25,  and 
0.3  c.c.  of  diluted  antigen  add  0.1  c.c.  of  fresh  heated  glanders  serum 
(known  to  yield  a  positive  reaction)  and  1  c.c.  of  complement  serum 
(1  : 20)  and  sufficient  salt  solution.  Incubate  for  one  hour  and  add  2 
units  of  hemolytic  amboceptor  and  corpuscles.  After  a  second  incuba- 
tion of  from  one  to  two  hours  that  tube  shoiving  just  complete  inhibition 
of  hemolysis  contains  the  antigenic  unit,  and  double  this  amount  may  be 
used  in  performing  the  main  test,  providing  that  it  is  still  one-half  or, 
better,  but  one-quarter  the  anticomplementary  unit. 

A  hemolytic  system  control  is  included  in  both  titrations,  and  in  the 
antigenic  titration  an  additional  control  on  the  serum,  to  determine 
whether  it  is  free  from  anticomplementary  action. 

The  dilution  here  advised  may  be  too  low;  if  this  is  the  case,  the 
titrations  must  be  repeated  with  the  antigen  diluted  1  :  50  or  1  : 100. 

The  Test. — The  external  jugular  vein  of  the  horse  is  punctured  with 
a  sterile  needle,  and  from  5  to  10  c.c.  of  blood  is  collected  in  a  centrifuge 
tube  or  other  glass  container,  which  preferably  should  be  sterile.  The 
clear  serum  is  heated  to  55°  C.  for  one-half  hour  just  before  the  tests  are 
conducted. 

Into  a  series  of  four  small  test-tubes  place  the  following  doses  of 
serum:  0.05,  0.1,  0.2,  and  0.2  c.c.  To  the  first  three  tubes  add  the 
proper  dose  of  antigen;  to  all  tubes  add  1  c.c.  of  complement  (1  :  20)  and 
sufficient  salt  solution  to  bring  the  total  volume  up  to  3  c.c. 

The  following  controls  should  be  included: 

1.  The  serum  control  on  each  serum  is  conducted  in  the  fourth  tube 
of  each  set. 

2.  A  known  positive  serum  should  be  tested  in  the  same  manner. 


COMPLEMENT-FIXATION   TEST    IN    CONTAGIOUS    ABORTION          513 

3.  A  known  negative  serum  should  be  tested  in  the  same  manner. 

4.  The  antigen  control,  which  at  this  stage  contains  the  dose  of 
antigen,  complement,  and  saline  solution. 

5.  The  hemolytic  control,  which  at  this  time  contains  but  1  c.c.  of 
complement  plus  saline  solution. 

6.  The  corpuscle  control,  containing  1  c.c.  of  corpuscle  suspension 
and  3  c.c.  of  saline  solution.    This  tube  should  be  plugged  with  cotton. 

All  tubes  are  gently  shaken  and  incubated  for  an  hour  in  a  water- 
bath  at  38°  C.  or  in  a  refrigerator  at  8°  to  12°  C.  for  four  to  six  hours  or 
over  night,  after  which  2  units  of  hemolytic  amboceptor  and  1  c.c.  of 
corpuscle  suspension  are  added  to  all  except  the  corpuscle  control. 
The  tubes  are  gently  shaken  and  reincubated  in  a  water-bath  for  an 
hour  or  two,  depending  upon  the  hemolysis  of  the  controls.  Primary 
incubation  in  the  refrigerator  is  especially  recommended. 

The  controls  are  first  inspected.  They  should  all  show  complete 
hemolysis,  except  the  first  three  tubes  of  the  positive  serum  series  and  the 
corpuscle  control.  Inhibition  of  hemolysis  in  the  first  three  tubes  of 
the  series  containing  the  unknown  serum  indicates  a  strong  positive 
reaction.  Complete  hemolysis  in  all  tubes  indicates  a  negative  reaction. 
Partial  hemolysis  in  the  first  three  tubes  indicates  a  partially  positive 
reaction.  If  the  serum  control  or  antigen  control  tubes  should  show  in- 
hibition of  hemolysis,  these  were  probably  anticomplementary  and  the 
test  should  be  repeated. 


COMPLEMENT-FIXATION  TEST  IN  CONTAGIOUS  ABORTION 
It  is  now  generally  conceded  among  veterinarians  that  the  Bacillus 
abortus  of  Bang  is  the  specific  cause  of  contagious  abortion  of  cows. 

Evidence  is  gradually  accumulating  to  show  that  an  organism  be- 
longing to  the  paratyphoid  group  is  frequently  the  cause  of  a  similar 
condition  among  mares  (Kilbourne  and  Smith,1  Liguierer,2  Liguierer 
andZabala;  Good;3  VanNeelsbergen;4  de  Jong;5  Meyer  and  Boerner)6. 
Meyer  and  Boerner,  who  have  studied  this  bacillus  with  particular  care, 
classify  it  with  the  paratyphoid-enteritidis  group  (Bacillus  aborti  equi). 

1  United  States  Department  of  Agriculture,  Bulletin  No.  3,  1893,  49  and  53. 

2  Rec.  Med.  veterinaire,  Ixxxii,  1905. 

3  Kentucky  Agriculture  Exper.  Station  Bulletin  No.  165,  1912. 

4  Tijdschrift,  v.  Veeartsenijk.,  xxiv,  1912. 

5  Archiv.  f.  Wissenshaftl.  u.  prak.  Tierheilkunde,  xxv,  1900. 

6  Jour.  Med.  Research,  1913,  xxix,  No.  2,  325. 

33 


514  THE    TECHNIC    OF    COMPLEMENT-FIXATION    HE  ACTIONS 

Veterinarians  are  generally  agreed  that  in  contagious  abortion  of 
cows  the  complement-fixation  test  is  highly  specific,  and  is  frequently  of 
considerable  value  in  establishing  a  diagnosis  (Meyer  and  Hardenburgh 
and  others). 

Meyer  and  Boerner  have  found  fixation  of  complement  to  occur  in 
contagious  abortion  of  mares  with  an  antigen  of  Bacillus  abortus  equi, 
and  recommend  the  test  as  diagnostic  aid  in  this  infection. 

Preparation  and  Standardization  of  Antigens. — The  antigen  of 
Bacillus  abortus  (Bang)  for  use  in  the  complement-fixation  test  in 
contagious  abortion  of  cows  is  prepared  by  cultivating  a  number  of 
strains  .of  the  bacillus,  which  have  been  trained  to  grow  aerobically, 
upon  slants  of  glycerin  agar  for  seventy-two  hours.  The  growths  are 
then  washed  off  with  sufficient  normal  saline  solution  containing  2  per 
cent,  phenol  to  yield  a  cloudy  emulsion.  Shake  briskly  in  order  to 
break  up  the  clumps  of  bacilli,  and  filter  through  paper.  Place  in  a 
refrigerator  for  several  days  to  complete  the  sterilization,  and  titrate 
the  anticomplementary  dose  each  time  before  the  main  test  is  conducted. 

The  antigen  may  also  be  prepared  by  cultivating  a  number  of  strains 
in  glycerin-serum  bouillon  for  five  or  six  weeks.  Centrifuge  thoroughly 
and  wash  the  bacilli  once  or  twice  with  normal  saline  solution,  to  remove 
all  traces  of  serum.  Dilute  the  washed  bacilli  with  sufficient  normal 
saline  solution  to  give  an  emulsion  equal  in  density  to  a  twenty-four- 
hour  bouillon  culture  of  Bacillus  coli,  and  add  0.4  per  cent,  of  phenol  as  a 
preservative. 

The  antigen  of  Bacillus  abortus  equi  for  making  the  complement- 
fixation  diagnosis  of  contagious  abortion  of  mares  is  prepared  of  eigh- 
teen- to  twenty-hour-old  glycerin  bouillon  cultures,  with  an  addition 
of  0.5  per  cent,  of  phenol.  These  antigens  are  less  anticomplementary 
than  shake  extracts,  and  keep  their  titer  unaltered  for  many  weeks 
(Meyer  and  Boerner).  They  may  also  be  used  for  making  the  mac- 
roscopic agglutination  test.  These  antigens  may  also  be  prepared 
after  the  method  used  in  the  preparation  of  gonococcus  antigen  (page 
506). 

The  anticomplementary  dose  is  determined  each  time,  and  one-half 
this  amount  is  used  in  performing  the  main  test.  The  technic  is  the 
same  as  that  employed  in  the  titration  of  glanders  antigen. 

The  tests  and  controls  are  conducted  with  descending  doses  of  fresh 
inactivated  serum  (0.05,  0.1,  and  0.2  c.c.),  in  exactly  the  same  manner 
as  the  glanders  reaction  is  performed. 


COMPLEMENT    FIXATION    IN    DOURINE  515 

COMPLEMENT  FIXATION  IN  DOURINE 

Dourine,  or  horse  syphilis,  is  a  specific  infectious  disease  of  the  horse 
and  ass,  transmitted  from  animal  to  animal  by  the  act  of  copulation, 
and  caused  by  the  Trypanosoma  equiperdum.  It  is  characterized  by  an 
irregular  incubation  period,  the  localization  of  the  early  symptoms  to 
the  .genital  organs,  and,  finally,  by  complete  paralysis  of  the  posterior 
extremities,  a  fatal  termination  ensuing  in  from  six  months  to  two  years. 

The  disease  is  especially  prevalent  among  horses  in  the  northwestern 
states,  and  may  occur  in  such  various  and  atypical  forms  as  to  render 
clinical  diagnosis  difficult. 

Complement-fixation  methods  of  diagnosis  have  been  tried  by  Pav- 
losvici,  Winkler  and  Wyschelersky,  Moller,  Watson,  Brown,  and  in  a 
large  series  of  cases  with  good  results  by  Moller,  Eichhorn,  and  Buck.1 
These  last-named  investigators  examined  8657  specimens  of  blood  from 
horses  in  Montana  and  North  and  South  Dakota,  and  of  these,  1076 
yielded  positive  reactions. 

In  most  of  these  experiments  the  results  were  corroborated  by  clinical 
and  pathologic  findings,  and  the  investigators  conclude  that  the  com- 
plement-fixation test  is  of  great  value,  especially  in  countries  where  only 
one  of  these  protozoan  diseases  exists. 

Preparation  and  Standardization  of  Antigen. — This  is  the  most 
difficult  part  of  the  technic,  because  the  trypanosome  is  not  readily 
grown  on  artificial  culture-media.  Watery,  alcoholic,  and  acetone 
extracts  of  various  organs  of  horses  dead  of  the  disease  do  not  yield 
satisfactory  antigens.  Since  the  reaction  is  a  group  reaction,  and 
dourine  is  the  only  trypanosome  infection  in  this  country,  Moller, 
Eichhorn,  and  Buck  selected  the  surra  organism  for  the  preparation  of 
antigen.  After  infecting  a  dog  and  at  the  height  of  infection  with- 
drawing 200  c.c.  of  blood  into  potassium  citrate  and  hemolyzing  with 
0.5  gram  of  saponin,  the  trypanosomes  were  secured  after  thorough 
centrifugalization  and  washed  three  times.  After  the  last  washing  the 
trypanosomes  were  emulsified  in  50  c.c.  of  salt  solution  and  preserved 
with  phenol.  This  antigen  yielded  highly  satisfactory  results,  but  the 
difficulty  of  preparing  it,  and  the  small  quantity  secured,  made  it 
necessary  that  another  method  be  used. 

An  extract  of  the  spleen  of  a  rat  just  dead  of  surra  was  found  to  yield 
a  satisfactory  antigen.  The  extract  does  not  keep  well,  and  must  be 
prepared  freshly  every  few  days  and  carefully  standardized.  Gray  or 

1  Amer.  Jour.  Veter.  Med.,  1913,  viii,  581. 


516  THE   TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

white  rats  are  infected  with  surra  by  injecting  0.2  c.c.  of  blood  from  a 
rabbit  with  this  disease.  If  a  large  number  of  tests  are  to  be  made,  the 
rats  should  be  so  infected  that  one  or  two  are  available  each  day  for  the 
preparation  of  the  antigen. 

The  spleen  from  a  rat  with  a  small  amount  of  salt  solution  added  is 
ground  in  a  mortar  until  a  pulpy  mass  results.  More  of  the  salt  solution 
is  added  from  time  to  time,  and  the  suspension  thus  obtained  is  filtered 
twice  through  a  double  layer  of  gauze  and  diluted  with  salt  solution  to 
40  c.c. 

The  anticomplementary  and  antigenic  doses  are  then  determined, 
and  the  extract  used  in  double  the  antigenic  unit,  providing  that  this 
amount  is  not  more  than  half  the  anticomplementary  dose.  If  a  posi- 
tive serum  from  an  infected  horse  is  not  available,  the  anticomplementary 
dose  may  be  determined  and  half  this  amount  used  in  conducting  the 
main  test. 

Anticomplementary  Titration. — Into  a  series  of  six  test-tubes  place 
increasing  amounts  of  antigen — 0.1,  0.2,  0.3,  0.4,  0.5,  and  0.6  c.c.;  add  1 
c.c.  of  complement  (1  :  20)  and  sufficient  salt  solution  to  bring  the  total 
volume  in  each  tube  up  to  2  c.c.  Incubate  for  one  hour  in  a  water-bath 
at  38°  C.  or,  preferably,  in  a  refrigerator  ot  8°  C.  for  six  hours  or 
over  night.  Add  2  units  of  antisheep  amboceptor  (determined  by  pre- 
liminary titration)  and  1  c.c.  of  2.5  per  cent,  sheep's  corpuscles.  Incu- 
bate for  one  hour  in  a  water-bath,  after  which  the  reading  is  made. 

The  Test. — The  serum  is  inactivated  and  used  in  dose  of  0.15  c.c., 
since  it  has  been  found  that  fixation  in  this  quantity  is  obtained  only 
with  serums  of  horses  affected  with  dourine.  In  some  instances  the 
serum  of  horses  has  reacted  in  doses  as  small  as  0.02  c.c.,  and  the  reac- 
.tion  may  be  conducted  with  increasing  doses  of  serum — 0.05,  0.1,  and 
0.15  c.c. — in  exactly  the  same  manner  as  when  the  glanders  reaction  is 
performed.  The  same  Controls  are  included. 


COMPLEMENT-FIXATION  TEST  IN  TYPHOID  FEVER 
This  was  one  of  the  original  diseases  in  which  Bordet  and  Gengou 
first  demonstrated  the  occurrence  of  complement  fixation.  Widal  and 
Lesourd  attempted  to  make  practical  application  of  this  method  in  the 
diagnosis  of  the  disease,  but  their  results  were  indifferent,  and  since  then 
numerous  writers  have  expressed  various  opinions  as  to  the  value  of  the 
test.  Garbat  has  secured  uniform  and  reliable  reactions  with  a  poly- 
valent antigen,  and  emphasizes  the  importance  of  this  factor. 


COMPLEMENT-FIXATION   TEST    IN   TUBERCULOSIS  517 

The  antigen  is  prepared  of  numerous  strains  of  typhoid  bacilli — the 
more  the  better.  Cultures  are  grown  on  slants  of  agar  for  forty-eight 
hours,  washed  off  with  small  quantities  of  sterile  distilled  water,  heated 
to  60°  C.  for  two  hours,  shaken  mechanically  for  twenty-four  hours,  and 
either  filtered  through  a  sterile  Berkefeld  filter  or  thoroughly  centrif- 
ugalized.  The  filtrate  is  preserved  with  0.5  per  cent,  phenol  and  used 
as  antigen. 

I  have  secured  good  results  by  removing  the  growths  with  small 
amounts  of  normal  salt  solution,  and  placing  them  in  a  shaking  flask, 
and  shaking  for  an  hour  to  break  up  the  clumps.  After  heating  to  60°  C. 
for  an  hour,  1  per  cent,  glycerin  and  0.5  phenol  are  added  as  preservatives, 
and  the  mixture  stored  away  in  ampules  containing  1  c.c.  each.  The 
emulsion  should  be  slightly  milky  in  appearance. 

The  antigen  is  diluted  1  : 10  or  1  :  20,  and  the  anticomplementary 
dose  determined  by  titration  before  the  main  tests  are  conducted.  The 
technic  of  the  reaction  is  exactly  similar  to  the  gonococcus  fixation  test. 

The  ease  with  which  the  Widal  reaction  is  performed  renders  it  the 
method  of  choice.  Nevertheless  the  complement-fixation  test  is  quite 
delicate,  and  will  frequently  aid,  where  the  agglutination  test  is  negative 
or  absent  and  in  making  the  differential  diagnosis  from  paratyphoid 
fever.  The  strongest  reactions  are  secured  late  in  the  disease. 


COMPLEMENT-FIXATION  TEST  IN  TUBERCULOSIS 
It  was  the  original  studies  in  complement  fixation  in  tuberculosis 
made  by  Wassermann  and  Bruch  that  later  induced  these  workers,  in 
cooperation  with  Neisser,  to  apply  the  method  to  the  diagnosis  of  syphilis. 
The  first  application  of  complement-fixation  in  tuberculosis  was 
made  by  Widal  and  LeSourd1  in  1901.  They  obtained  deviation  of 
complement  in  certain  cases  of  tuberculosis,  using  as  antigen  homoge- 
neous emulsions  of  tubercle  bacilli  of  the  Arloing-Courmont  strain.  In 
1903  Bordet  and  Gengou2  demonstrated  the  presence  of  antibody 
capable  of  uniting  with  tubercle  bacilli  and  fixing  complement  in  the 
sera  of  tuberculous  animals.  Wassermann  and  Bruck3  in  1906  demon- 
strated the  presence  of  an  antibody  to  tuberculin  in  patients  treated 
with  tuberculin,  but  they  examined  only  13  cases  of  pulmonary  tuber- 
culosis. Caulfield4  in  1911  examined  104  cases  of  pulmonary  tubercu- 

1  Cited  by  Sherman  and  Miller,  Edinburgh  Med.  Jour.,  1913,  10,  81. 

2  Compt.  rend.  Acad.  de  Sc.,  1903,  137,  351. 

3  Deutsch.  med.  Wchnschr.,  1906,  32,  449. 

4  Jour.  Med.  Research,  1911,  24,  122. 


518  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

losis  with  bacillary  emulsion  as  antigen  and  obtained  33  per  cent. 
Turban  I  cases,  70  per  cent.  Turban  II,  and  62  per  cent.  Turban  III 
positive  results.  Laird1  (1912)  out  of  84  tests  in  34  cases  obtained  24 
positives  in  4  cases,  using  watery  emulsion  of  tubercle  bacilli  (which 
,he  does  not  describe);  his  results  were  inconclusive.  Hammer,2  using 
O.  T.  and  extracted  tuberculous  nodules,  obtained  97  per  cent,  positive 
results  in  46  tuberculous  cases.  Calmette  and  Massol,3  using  prepara- 
tions made  from  tubercle  bacilli  by  extracting  with  water  and  peptone, 
obtained  in  134  cases  92.5  per  cent,  fixation.  Fraser4  (1913),  testing  a 
large  variety  of  antigens,  found  that  living  bacilli  gave  no  fixation  in 
96.6  per  cent,  of  normal  individuals,  but  gave  positive  reactions  in 
42.3  per  cent,  of  tuberculous  individuals.  She  states  that  the  most 
reliable  antigen  is  prepared  from  living  human  bacilli,  and  that  diag- 
nostically  the  complement-fixation  test  with  living  bacilli  is  of  more 
value  from  the  standpoint  of  positive  results  than  any  other  reaction 
discovered  to  date.  She  believes  the  absence  of  antibodies  accounts 
for  the  low  percentage  of  results  obtained.  Dudgeon,  Meek,  and  Weir5 
also  tested  a  large  number  of  antigens,  and  in  102  untreated  cases 
obtained  86  positive  results,  while  all  cases  which  had  been  treated 
with  tuberculin  gave  positive  results.  Products  of  the  bacilli  themselves 
were  found  to  be  the  most  satisfactory  as  antigen.  With  an  alcoholic 
antigen6  prepared  from  tubercle  bacilli  they  obtained  from  a  total  of 
234  cases,  209  (89.3  per  cent.)  positives,  194  of  these  on  first  examination, 
11  (of  the  15  negative)  on  second  examination,  and  4  more  on  third 
examination.  Besredka7  (1913)  prepared  an  antigen  by  growing 
tubercle  bacilli  on  egg  broth,  heating  it,  and  filtering.  W'ith  this  antigen 
Bronfenbrenner8  (1914)  obtained  a  very  high  percentage  of  positive 
results,  93.8  per  cent,  in  active  cases,  and  55.5  per  cent,  in  convalescents, 
while  suspected  cases  gave  75  per  cent,  and  syphilitic  sera  24  per  cent, 
positive  reactions.  Inman9  and  Kuss,  Leredde  and  Rubenstein10  found 
this  antigen  non-specific.  Mclntosh,  Fildes,  and  Radcliffe11  (1914) 
also  justly  criticized  Besredka's  antigen,  and  concluded,  after  testing  a 
large  number  of  antigens,  that  the  living  bacillary  emulsion  was  best, 

1  Jour.  Med.  Research,  1912,  27,  163. 

2  Miinchen.  med.  Wehnschr.,  1912,  59,  1750. 

3  Compt.  rend.  Soc.  de  biol.,  1912,  73,  120. 
4Ztschr.  f.  Immunitatsf.,  1913,  20,  291. 

6  The  Lancet,  1913,  184,  19.  6  Jour.  Hyg.,  1914,  14,  52,  72. 

7  Compt.  rend.  Acad.  d.  sc.,  1913,  156,  1633. 

8  Arch.  Int.  Med.,  1914,  14,  786. 

9  Compt.  rend.  Soc.  de  biol.,  1914,  76,  251. 

10  Ibid.,  244.  "  The  Lancet,  1914,  185,  485. 


COMPLEMENT-FIXATION    TEST    IN    TUBERCULOSIS  519 

yielding  76.7  per  cent,  positive  results  in  43  definite  cases  of  phthisis, 
80.7  per  cent,  in  surgical  tuberculosis,  and  37.5  per  cent,  in  glandular 
tuberculosis.  Of  87  normal  individuals,  only  3  gave  positive  reactions 
(2  of  these  were  lepers  and  1  had  Addison's  disease.)  Negative  reactions 
were  obtained  in  18  syphilitic  patients.  They  look  upon  a  positive 
reaction  as  indicative  of  active  tuberculosis.  Stimson1  (1915),  who 
gives  a  fairly  exhaustive  table  of  the  recent  literature,  reports  a  small 
number  of  cases  in  which  a  variety  of  antigens  were  used,  but  his  re- 
sults were  inconclusive.  Craig2  (1915)  reports  the  results  of  examination 
of  166  cases  of  pulmonary  tuberculosis  in  which  he  employed  as  antigen 
an  alcoholic  extract  of  several  strains  of  human  tubercle  bacilli  which 
had  been  grown  on  a  liquid  medium  of  alkaline  broth  containing  egg; 
96.2  per  cent,  positive  results  were  obtained  in  active  cases  and  66.1  per 
cent,  positive  in  inactive  cases.  One  hundred  and  fifty  cases  of  syphilis 
gave  only  2  positive  reactions,  and  these,  on  further  examination,  re- 
vealed lesions  in  the  lungs.  One  hundred  other  diseases  examined  all 
gave  negative  results.  In  a  later  report  Craig,  using  his  same  antigen 
of  a  filtrate  of  an  alcoholic  extract  of  several  strains  of  human  tubercle 
bacillus  plus  the  culture-medium  in  which  the  bacilli  had  been  grown, 
has  reported  further  favorable  results  in  complement-fixation  in  tuber- 
culosis. Miller  and  Zinsser3  have  described  a  simple  antigen  prepared 
by  grinding  living  or  dead  tubercle  bacilli  with  dry  table  salt,  and  then 
adding  distilled  water  up  to  isotonicity.  With  this  antigen  they  have 
reported  excellent  results;  Miller4  found  the  reaction  practically  always 
positive  in  active  tuberculosis  with  negative  reactions  in  non-tuberculous 
syphilitic  and  normal  persons.  In  my  own  studies  in  co-operation  with 
Montgomery,  prepared  after  Miller's  method,  yielded  positive  reactions 
only  with  the  sera  of  tuberculous  persons,  but  the  percentage  of  positive 
reactions  was  much  less  than  reported.  Corber,5  employing  an  autolysate 
of  tubercle  bacilli  as  antigen,  found  30  per  cent,  positive  reactions  with 
clinically  definite  cases  of  tuberculosis  both  active  and  inactive.  Active 
cases  gave  a  higher  percentage  of  positive  results  than  inactive  cases. 
Eichhorn  and  Blumberg,6  using  Besredka's  antigen  and  the  sera  of  tuber- 
culous persons  and  the  lower  animals,  found  the  complement-fixation 
test  less  reliable  than  the  subcutaneous  tuberculin  test  in  the  diagnosis 

1  Bull.  Hyg.  Lab.  U.  S.  P.  H.  and  M.  H.  S.,  1915,  No.  101,  7. 

2  Amer.  Jour.  Med.  Sci.,  1915,  150,  781;  Jour.  Amer.  Med.  Assoc.,  1917,  Ixviii, 
773. 

3  Proc.  Soc.  Exper.  Biol.  and  Med.,  1916,  xiii,  134. 

4  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  1519. 

5  Jour.  Infect.  Dis.,  1916,  19,  315.        6  Jour.  Agricult.  Research.,  1917,  viii,  1. 


520  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

of  tuberculosis  in  cattle  and  not  practical  for  general  diagnostic  purposes. 
Lucke1  found  that  the  wax  of  tubercle  bacilli  possessed  no  antigenic 
properties;  Wood,  Bushnell,  and  Maddux2  have  observed  a  large  per- 
centage of  apparently  specific  reactions  with  the  partial  antigens  of 
Deyke  and  Much.3 

Preparation  and  Standardization  of  Antigen. — It  is  apparent,  there- 
fore, that  the  question  of  proper  antigen  is  of  paramount  importance  in 
complement  fixation  in  tuberculosis.  It  would  appear  that  Besredka's 
and  Craig's  antigens  and  suspensions  of  living  tubercle  bacilli  are  of 
definite  value.  The  main  objections  to  the  bacillary  emulsion  are  the 
small  difference  between  the  antigenic  and  anticomplementary  units, 
the  turbidity,  and  fairly  high  percentage  of  non-specific  reactions.  Eich- 
horn  and  Blumberg  have  given  the  following  directions  for  the  prep- 
aration of  a  modified  Besredka  antigen: 

Twenty  c.c.  each  of  the  white  and  the  yolk  of  an  egg  were  thoroughly 
beaten  in  an  automatic  egg-beater,  and  to  the  whipped  material  a  solu- 
tion of  Liebig's  meat  extract  (3  : 1000)  in  distilled  water  is  gradually 
added  while  the  mixture  was  continuously  beaten.  A  sufficient  meat- 
extract  infusion  is  used  to  make  up  the  whole  to  1000  c.c.  The 
emulsion  is  strained  through  cotton  and  heated  to  the  boiling-point, 
then  strained  again,  and  after  the  addition  of  0.5  per  cent,  of  sodium 
chlorid  is  carefully  neutralized,  heated  again  and  strained,  and  neutral- 
ized if  necessary.  To  the  neutral  medium  sufficient  normal  sodium 
hydroxid  -  solution  is  added  to  make  the  medium  of  0.2  alkalinity. 
This  medium,  to  which  neither  peptone  nor  glycerin  was  added,  was 
then  autoclaved  at  115°  C.  for  twenty  minutes,  and  after  cooling  is 
kept  at  37°  C.  for  forty-eight  hours  for  observation  as  to  sterility. 

Whatever  antigen  is  employed  it  must  be  carefully  titrated  for  the 
anticomplementary  unit  each  time  before  the  main  tests  are  conducted. 
I  conduct  these  titrations  in  the  same  manner  as  described  under  the 
gonococcus  complement-fixation  test  and  use  one-third  the  anticomple- 
mentary unit  as  the  dose  for  the  main  tests. 

The  Test. — These  may  be  conducted  with  a  0.2  c.c.  dose  of  serum 
or  with  three  graded  doses,  as  0.05,  0.1,  and  0.2  c.c.,  as  described  in  the 
gonococcus  complement-fixation  test.  I  have  generally  employed  the 
latter  with  a  primary  incubation  of  six  hours  or  over  night  in  the  refrig- 
erator at  8°  C.  or  one  hour  in  the  water-bath  at  38°  C. 

1  Jour.  Immunol.,  1916,  1,  457.  2  Jour.  Immunol.,  1917,  2,  301. 

3  Med.  Klinik,  1908,  4,  1540;  Munch,  med.  Wchnschr.,  1909,  56,  1895;  ibid., 
1909,  56,  1825;  Beitr.  z.  klin.  Tuberk.,  1910,  15,  277;  ibid.,  1911,  20,  343. 


COMPLEMENT    FIXATION    IN    PERTUSSIS  521 

Practical  Value  of  the  Complement-fixation  Test  in  Tuberculosis. — 
It  is  still  too  early  to  express  an  accurate  opinion  of  the  practical  value 
of  this  test;  an  analysis  of  numerous  reports  and  my  own  studies  appar- 
ently warrants  the  following  statements: 

1.  A  definitely  positive  reaction  taken  in  conjunction  with  other 
findings  makes  the  diagnosis  of  tuberculosis  certain  and  may  be  of 
distinct  aid  in  the  diagnosis  of  early  tuberculosis. 

2.  From  the  standpoint  of  differential  diagnosis  the  test  is  of  value 
when  positive  as  indicating  tuberculosis  against  other  diseases  of  the 
lungs,  as  carcinoma,  syphilis,  abscess,  empyema,  etc. 

3.  As  the  reaction  is  likely  to  be  positive  only  in  active  cases,  the 
complement-fixation  test  is  superior  to  other  biologic  tests  for  active 
tuberculosis,  as,  for  example,  the  skin  test,  which  is  likely  to  be  positive 
in  persons  with  quiescent  lesions. 

4.  A  persistently  and  strongly  positive  reaction  probably  indicates 
the  presence  of  active  tuberculosis. 

5.  The  strength  of  a  positive  reaction  appears  to  bear  some  relation 
to  the  severity  of  the  disease,  and  reactions  becoming  gradually  weaker 
until  negative  have  been  frequently  noted  with  clinical  improvement 
and  "cure." 

COMPLEMENT  FIXATION  IN  PERTUSSIS 

Investigations  on  complement  fixation  with  the  bacillus  of  pertussis 
has  given  varied  results,  perhaps  due  in  part  to  a  variation  in  the  cul- 
tures employed  and  the  methods  used  to  prepare  the  antigens. 

Bordet  and  Gengou1  used  suspensions  in  salt  -solution  of  growths 
on  solid  media.  Arnheim2  found  negative  results  with  antigens  of  both 
the  Bordet-Gengou  bacillus  of  pertussis  and  the  influenza  bacillus. 
Wollstein3  used  three  forms  of  antigen:  suspensions  of  the  bacilli  in 
salt  solution;  extracts  of  bacilli  made  by  suspending  the  growths  of 
three  blood-agar  slants  in  5  c.c.  of  salt  solution  and  shaking  for  twenty- 
four  hours  in  thermostat;  and  extracts  of  tissue  obtained  from  patients 
dying  from  pertussis.  Friedlander  and  Wagner4  used  live  bacteria  and 
fresh  serum,  and  considered  this  innovation  of  great  importance.  The 
different  hemolytic  systems  employed  may  also  have  had  some  bearing 
on  the  failure  to  obtain  identical  results. 

Bordet  and  Gengou  obtained  complement  fixation  in  all  of  their 

1  Ann.  de  1'Inst.  Pasteur,  1906,  20,  731. 

2  Berl.  klin.  Wchnschr.,  1908,  xlv,  1453;  Arch.  f.  Kinderhl.,  1909,  1,  295. 

3  Jour.  Exper.  Med.,  1909,  11,  p.  41. 

4  Amer.  Jour.  Dis.  of  Children,  1914,  8,  p.  134. 


522  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

cases  of  pertussis.  They  used  0.1  c.c.  to  0.3  c.c.  of  the  human  serum 
heated  to  56°  C.,  and  0.05  c.c.  to  0.01  c.c.  of  fresh  guinea-pig  serum 
as  complement.  The  amboceptor  was  the  serum  of  rabbits  previously 
injected  with  sheep  corpuscles.  The  antigen  was  an  emulsion  of  the 
bacillus  of  pertussis  in  salt  solution,  the  growth  being  twenty-four 
hours  old. 

Arnheim1  obtained  complement  fixation  in  6  of  12  cases  of  per- 
tussis. Wollstein  examined  the  serum  from  9  patients  with  pertussis 
and  in  no  instance  did  she  obtain  complement  fixation,  using  the 
three  forms  of  antigen  just  mentioned  in  all  cases.  The  quantities 
of  complement  and  amboceptor  were  about  the  same  as  those  used  by 
Bordet  and  Gengou. 

Gengou  and  Brunard2  describe  3  cases  in  which  they  determined 
the  specific  pertussis  character  of  the  infection  by  complement  fixation. 

In  1911  Bordet  and  Gengou3  reported  certain  atypical  cases  diagnosed 
as  pertussis  by  means  of  complement  fixation.  A  little  later  Bordet4 
concluded  that  the  power  to  fix  complement  is  not  found  early,  and  does 
not  become  marked  until  near  the  end  of  the  disease. 

St.  Bacher  and  Menschikoff5  report  27  cases  of  pertussis  in  all 
stages  in  which  attempts  were  made  to  obtain  complement  fixation, 
without  success  in  a  single  case.  Only  after  vaccines  of  pure  cultures 
of  the  Bacillus  pertussis  were  given  did  fixation  occur.  Their  antigen 
was  an  emulsion  of  the  bacillus  of  pertussis  in  salt  solution,  while  0.4  c.c. 
of  a  1  : 10  dilution  of  guinea-pig  serum  served  as  complement,  and 
0.5  c.c.  of  a  1  : 150  dilution  of  serum  of  a  rabbit  previously  injected 
with  sheep  corpuscles  served  as  amboceptor. 

Delcourt6  obtained  complement  fixation  in  6  cases  of  pertussis. 
Poleff7  gives  a  resume  of  the  results  of  five  investigators.  In  5  cases 
only  was  there  complement  fixation  and  in  31  cases  no  fixation  was 
obtained.  He  himself  reports  10  cases  in  which  he  did  not  obtain 
fixation. 

Hess8  tested  10  cases  and  concluded  "that  results  would  seem  to 
show  that  this  reaction  is  present  for  some  months  after  cessation  of 
all  symptoms." 

1  Berl.  klin.  Wchnschr.,  1908,  14,  p.  1453. 

2  Bull.  Adad.  de  med.  Belg.,  1910,  24,  p.  329. 

3  Centralbl.  f.  Bakteriol.,  I,  O,  1911,  58,  p.  573. 

4  Ibid.,  1912,  66,  p.  275.  •  Ibid.,  61,  p.  218. 

6  Presse  med.  Belg.,  1912,  64,  p.  19. 

7  Centralbl.  f.  Bakteriol.,  I,  O.,  1913,  69,  p.  23. 

8  Jour.  Amer.  Med.  Assoc.,  1914,  63,  p.  1007 


COMPLEMENT    FIXATION    IN    PERTUSSIS  523 

Recently  Anna  Wessels  Williams1  came  to  the  conclusion  that  the 
test  with  serum  of  human  beings  is  not  as  clear  cut  as  it  seems  to  be 
with  serum  of  animals  which  have  been  injected  with  the  bacillus  of 
pertussis. 

Renaux,2  using  the  Bacillus  pertussis  for  antigen,  examined  73  sera 
for  complement  fixation,  32  cases  of  which  were  known  cases  of  pertussis. 
Of  the  cases  of  pertussis,  he  obtained  complement  fixation  in  23.  Of 
the  9  negative  cases,  the  serum  had  been  obtained  early  during  the 
disease,  and  3  of  these  gave  positive  complement  fixation  when  the 
attack  had  lasted  about  four  weeks.  No  further  examination  was  made 
on  the  remaining  6  cases.  His  results  seem  to  show  that  fixation  appears 
about  three  or  four  weeks  after  the  appearance  of  the  whoop. 

In  18  cases  of  pertussis  Friedlander  and  Wagner3  obtained  comple- 
ment fixation  in  each  case.  They  used  fresh  serum  of  pertussis  patients 
and  living  bacteria.  The  Noguchi  hemolytic  system  was  used  throughout 
their  work  on  account  of  the  small  quantity  of  serum  necessary.  They 
claim  that  the  diagnosis  of  pertussis  can  be  made  with  certainty  in  the 
catarrhal  stage  by  means  of  complement  fixation.  In  a  more  recent 
article  Freidlander4  reports  further  results.  He  obtained  fixation  in 
13  of  14  cases  in  the  catarrhal  stage  before  the  characteristic  whoop 
had  appeared.  In  one  case  there  was  fixation  three  weeks  before  the 
first  whoop.  Winholt5  has  reported  positive  reactions  in  pertussis 
with  polyvalent  antigens  prepared  by  cultivating  the  bacilli  on  potato- 
glycerin  blood-agar,  suspending  in  normal  salt  solution,  and  heating 
to  56°  C.  for  thirty  minutes.  Olmstead  and  Povitsky6  have  been  able 
to  differentiate  between  Bacillis  pertussis  and  B.  influenza  by  means 
of  the  complement-fixation  tests  employing  rabbit  immune  sera. 

Preparation  and  Standardization  of  the  Antigen. — Antigens  may  be 
prepared  in  the  same  manner  as  gonococcus  antigen  (page  505)  and 
should  be  polyvalent.  The  antigen  should  always  be  titrated  for  the 
anticomplementary  unit  as  described  in  the  gonococcus  complement- 
fixation  test,  and  used  in  a  dose  corresponding  to  one-third  the  anti- 
complementary  unit. 

The  Test. — This  may  be  conducted  with  0.2  c.c.  of  serum  or  with 
graded  doses — 0.05,  0.1,  and  0.2  c.c. — as  described  under  the  gonococcus 

1  Arch.  Pediat.,  1914,  31,  p.  567. 

2  Centralbl.  f.  Bakteriol.,  I.  O.,  1914,  75,  p.  197. 

3  Amer.  Jour.  Dis.  of  Children,  1914,  8,  p.  134. 

4  The  Lancet-Clinic,  1915,  113,  p.  8. 
6  Jour.  Infect.  Dis.,  1915,  16,  389. 

6  Jour.  Med.  Research,  1916,  33,  379. 


524  THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

test.  The  serum  should  be  inactivated,  as  active  serum  is  likely  to 
yield  non-specific  reactions.  The  primary  incubation  for  complement 
fixation  should  be  one  hour  at  38°  C.  in  the  water-bath  or  six  hours  in 
the  refrigerator  at  8°  C. 

Practical  Value  of  the  Test. — Positive  reactions  are  not  usually  ob- 
tained until  the  paroxysmal  stage  is  reached,  when  about  50  per  cent, 
of  sera  react  positively  (Park).  For  this  reason  the  complement- 
fixation  test  possesses  little  value  in  early  diagnosis.  It  is  at  times  of 
value  in  the  diagnosis  of  atypical  cases. 

With  rabbit  immune  sera  the  test  has  proved  of  value  in  the  study 
and  differentiation  of  microorganisms  resembling  Bacillis  pertussis. 

THE   COMPLEMENT-FIXATION   TEST   IN   THE   STANDARDIZATION   OF 

IMMUNE  SERUMS 

The  technic  of  complement  fixation  has  also  been  employed  as  one 
means  in  effecting  the  standardization  of  antimeningococci  and  anti- 
gonococcic  serums.  Since,  however,  the  amount  of  complement-fixing 
amboceptors  in  a  serum  is  no  index  to  its  therapeutic  and  prophylactic 
value,  a  measure  of  this  one  factor  is  not  a  reliable  standard. 

The  technic  consists  in  preparing  the  antigen  and  in  determining  its 
anticomplementary  dose.  Whatever  this  is,  one-half  to  one-quarter 
this  amount  is  added  to  increasing  quantities  of  heated  immune  serum, 
ranging  from  0.001  to  0.1  c.c.  Complement  and  saline  solution  are 
added,  and  after  incubating  one  hour  at  37°  C.,  the  amount  of  comple- 
ment fixation  is  determined  by  adding  hemolytic  amboceptors  and  cor- 
puscles. 

While  this  titration  is  one  measure  of  the  reaction  of  the  animal  used 
in  the  immunization,  better  evidence  of  the  therapeutic  value  of  the 
serum  is  obtained  by  determining  the  content  in  bacteriotropins,  by 
testing  the  serum  with  the  antigen  in  susceptible  animals,  or  by  a  com- 
bination of  all  methods. 

THE  COMPLEMENT-FIXATION  TEST  IN  ECHINOCOCCUS  DISEASE 

Several  investigators  have  employed  a  complement-fixation  technic 

in  the  diagnosis  of  echinococcus  disease  of  the  liver.     Ghedini1  found 

this  test  of  value,  and  he  also  reports  specific  reactions  with  the  sera  of 

persons  infested  with  Ankylostoma  duodenales  and  Ascaris   lumbri- 

1  Gaz.  degli  Ospedali  e  delle  Cliniche,  1906,  27,  p.  1616;  1907,  28,  p.  53. 


COMPLEMENT-FIXATION    TEST    IN    ECHINOCOCCUS    DISEASE         525 

coides.1  Weinberg,2  Jiani,3  Israel,4  and  Henius5  report  favorable  and 
specific  reactions  in  echinococcus  disease  with  aqueous  or  alcoholic 
extracts  of  cyst  fluid,  or  both.  Kurt  Meyer6  found  these  reactions  non- 
specific, in  that  the  serum  of  a  person  infested  with  echinococcus  showed 
complement-absorption  with  antigens  of  Tsenia  solium  and  T.  saginata, 
and  vice  versa.  Branes7  found  that  alcoholic  extracts  of  echinococcus 
cyst  fluid  showed  complement-absorption  with  the  syphilis  antibody 
as  well  as  with  that  of  echinococcus  disease,  and  this  observation  has 
been  generally  confirmed.  Thomsen  and  Magnusson8  in  a  study  of  12 
cases  of  echinococcus  disease  found  that  the  sera  of  10  reacted  positively; 
the  sera  of  55  control  cases  (32  of  which  reacted  positively  to  the  Wasser- 
mann  reaction)  all  were  negative  except  one.  These  authors  also  report 
favorabty  on  the  specificity  of  the  reaction:  the  sera  of  10  persons  in- 
fected with  Tsenia  saginata,  of  2  with  T.  solium,  and  of  1  with  Bothrio- 
cephalus  latus,  all  were  negative  with  echinococcus  antigen. 

In  so  far  as  echinococcus  disease  of  the  liver  is  concerned,  a  review 
of  the  literature  shows  a  general  consensus  of  opinion  that  antibodies 
are  present  in  the  sera  of  the  majority  of  diseased  persons  and  animals 
and  that  these  may  be  detected  by  means  of  a  complement-fixation  test. 
There  is,  however,  a  division  of  opinion  in  regard  to  the  specificity  of 
the  reaction  and  its  practical  value  in  diagnosis. 

Much  less  work  has  been  reported  on  complement  fixation  with  sera 
of  persons  infested  with  such  parasites  as  Tsenia  saginata,  Ascaris 
lumbricoides,  etc.  Miss  Trist  and  I9  have  observed  positive  reactions 
with  sera  of  dogs  infested  with  various  parasites  and  salt  solution 
extracts  of  the  respective  worms.  The  method  is  worthy  of  trial  in 
the  diagnosis  of  infestments  of  persons  with  the  various  intestinal 
parasites. 

The  antigen  is  best  prepared  of  the  fresh  fluid  of  an  echinococcus 
cyst  of  man  or  sheep.  It  should  be  filtered,  if  necessary,  preserved  with 

1  Gaz.  degli  Ospedali  e  delle  Cliniche,  1907,  28,  p.  476. 

2  Ann.  de  PInst.  Pasteur,  1909,  23,  p.  472  (in  which  references  are  given  to  his 
previous  work  in  this  field). 

3  Wien.  klin.  Wchnschr.,  1909,  22,  p.  1439  (in  which  the  author  gives  a  good  bib- 
liography of  earlier  literature). 

4  Ztschr.  f.  Hyg.  u.  Infektionskrankh.,  1910,  66,  p.  487. 

5  Deutsch.  med.  Wchnschr.,  1911,  37,  p.  1212. 

6  Berl.  klin.  Wchnschr.,  1910,  47,  p.  1316. 

7  Miinchen.  med.  Wchnschr.,  1911,  58,  p.  1073. 
s  Berl.  klin.  Wchnschr.,  1912,  49,  p.  1183. 

9  Jour.  Infect.  Dis.,  1916,  18,  88. 


526  THE    TECHNIC    OF    COMPLEMENT-FIXATION    REACTIONS 

0.5  per  cent,  phenol,  and  kept  constantly  at  a  low  temperature.  It  is 
highly  important  not  to  use  the  antigen  in  an  anticomplementary  dose, 
hence  it  should  be  titrated  before  each  test  is  made. 

Anticomplementary  Titration. — Dilute  the  fluid  1  : 10  with  normal 
saline  solution,  and  into  a  series  of  eight  small  test-tubes  place  increasing 
amounts  as  follows:  0.1,  0.2,  0.4,  0.6,  0.8,  1,  2,  and  3  c.c.  Add  1  c.c.  of 
complement  (1  :  20)  and  sufficient  saline  solution  to  bring  the  total 
volume  in  each  tube  up  to  4  c.c.  Shake  gently,  incubate  for  one  hour, 
and  then  add  2  units  of  antisheep  amboceptor  (previously  titrated) 
and  corpuscles.  Reincubate  for  one  to  two  hours;  the  tube  showing 
beginning  inhibition  of  hemolysis  contains  the  anticomplementary  dose, 
and  in  performing  the  main  test,  one-half  to  one-quarter  this  amount 
may  be  used. 

If  titration  wTith  diluted  fluid  does  not  give  the  anticomplementary 
dose,  the  titration  should  be  repeated  with  undiluted  fluid. 

Alcoholic  extracts  of  daughter  cysts,  the  hydatid  wall,  or  the  fluid 
have  been  found  to  give  positive  reactions  in  syphilis  (Israel,  Brauer, 
Henins),  and  are,  therefore,  unsatisfactory. 

Thomsen  and  Magnussen  speak  favorably  of  antigen  paper  infil- 
trated with  cyst  fluid  and  titrated. 

The  Test. — The  patient's  serum  should  be  fresh,  and  should  be  heated 
to  55°  C.  for  half  an  hour.  Into  a  series  of  five  test-tubes  place  0.025, 
0.05,  0.1,  0.2,  and  0.2  c.c.  of  serum.  To  each  of  the  first  four  tubes  add 
one-half  the  anticomplementary  dose  of  antigen;  to  all  the  tubes  add 
1  c.c.  of  complement  (1  :  20)  and  sufficient  saline  solution  to  bring  the 
total  volume  up  to  3  c.c. 

The  fifth  tube  is  the  serum  control;  an  antigen,  hemolytic  system, 
and  corpuscle  control  should  be  included,  as  is  usual  in  complement- 
fixation  tests.  A  normal  serum  may  be  included,  and,  if  possible,  a 
known  positive  serum. 

After  incubating  for  an  hour  at  37°  C.,  add  2  units  of  hemorytic 
amboceptor  and  1  c.c.  of  the  corpuscle  suspension  to  each  tube.  Re- 
incubate  for  an  hour  or  longer,  depending  upon  the  hemolysis  of  the 
controls.  Marked  or  complete  inhibition  of  hemolysis  in  the  fourth 
tube  (0.2  c.c.  patient's  serum),  with  lesser  degrees  of  inhibition  in  the 
other  tubes  of  the  series,  indicates  a  positive  reaction.  Larger  doses  of 
patient's  serum  should  not  be  used  on  account  of  the  probability  of  non- 
specific complement  fixation. 


PROTEIN   DIFFERENTIATION    BY    COMPLEMENT    FIXATION  527 

PROTEIN  DIFFERENTIATION  BY  COMPLEMENT  FIXATION 
The  Determination  of  an  Antigen  by  Complement  Fixation. — In  the 

tests  hitherto  considered  the  antigens  were  known,  and  the  suspected 
antibodies  sought  for  in  the  blood-serum  or  other  body-fluid.  In  making 
the  reactions  it  was  necessary  to  bring  the  serum  to  be  tested  into  con- 
tact with  the  antigen  specific  for  the  suspected  antibody,  in  the  presence 
of  complement,  and  at  a  suitable  temperature.  At  the  end  of  an  hour 
the  mixture  was  tested  for  free  complement  by  adding  hemolytic  ambo- 
ceptor  and  red  blood-corpuscles.  This  order  may  be  reversed,  and  with 
a  known  antibody  the  suspected  antigen  may  be  detected.  The  anti- 
gen to  be  detected,  as  in  a  solution  of  blood  or  bacterial  extract,  is 
brought  into  contact  with  its  specific  antibody  in  the  presence  of  com- 
plement. At  the  end  of  an  hour,  at  a  suitable  temperature,  the  mixture 
is  tested  as  previously  for  free  complement,  by  adding  corpuscles  and 
hemolytic  amboceptors.  Under  proper  conditions  complement  fixation 
would  indicate  a  specific  reaction  between  the  antibody  and  its  antigen, 
and  thus  serve  to  identify  the  latter. 

This  method  has  clinical  applications  similar  to  those  in  which  the 
precipitin  reaction  is  used : 

1.  In  the  differentiation  of  blood-stains  a  solution  of  the  stain  con- 
stitutes the  unknown  antigen.     By  furnishing  a  known  antiserum  the 
antigen  is  detected,  i.  e.,  the  animal  from  which  the  blood  was  derived 
is  ascertained. 

2.  In  the  recognition  and  differentiation  of  meats. 

3.  In  the  detection  of  bacterial  antigens  in  the  blood-serum  of 
patients,  or  with  highly  immune  serums  an  unknown  bacterial  antigen 
may  be  identified  and  the  test  employed  as  a  means  of  differentiation 
among  bacterial  species. 

4.  Similar  applications  of  the  test  may  be  made  in  the  differentiation 
of  milks,  seminal  stains,  and  other  albuminous  substances. 

As  compared  with  precipitin  reactions,  the  complement-fixation  test 
is  probably  more  delicate  and  reliable  and  easier  of  interpretation.  The 
technic  of  the  latter  method  is,  however,  more  complicated,  and  the 
liability  to  error  is  greater  unless  the  principles  of  complement  fixation 
in  general  are  thoroughly  understood  and  the  importance  of  quantitative 
factors  is  appreciated. 

1.  Complement  Fixation  for  the  Identification  of  Blood-stains. — The 
application  of  the  technic  of  complement  fixation  to  the  determination 
of  specific  protein  antigen,  such  as  human  or  animal  blood,  was  demon- 


528  THE   TECHNIC   OF   COMPLEMENT-FIXATION   REACTIONS 

strated  by  Gengou  in  1902.  The  principles  worked  out  by  him  were 
extensively  studied  and  practically  applied  by  Neisser  and  Sachs  in  the 
forensic  differentiation  of  animal  proteins. 

Hemolytic  System. — Complement  is  furnished  by  the  fresh  serum  of 
a  guinea-pig  diluted  1  :  20  and  used  in  dose  of  1  c.c.  (  =  0.05  c.c.  serum); 
washed  sheep's  corpuscles  are  made  up  in  a  2.5  per  cent,  suspension  and 
used  in  dose  of  1  c.c.;  antisheep  amboceptor  should  be  highly  potent, 
and  is  titrated  after  the  method  previously  given  (p.  399).  In  the 
following  titrations,  and  in  conducting  the  main  test  the  hemolytic 
amboceptor  is  used  in  an  amount  equal  to  2  units. 

Specific  Antiserum. — This  is  obtained  from  a  rabbit  immunized 
with  the  protein  for  which  the  test  is  to  be  made,  namely,  human  or 
animal  blood-serum.  In  forensic  tests  it  may  be  necessary  to  prepare 
a  number  of  these  antiserums  with  the  serums  of  man  and  the  ordinary 
domestic  animals.  The  technic  of  immunization  is  the  same  as  that 
employed  for  the  preparation  of  precipitins  (p.  70).  An  antiserum  for 
forensic  tests  must  be  sufficiently  potent  to  fix  complement  with  0.0001 
c.c.  of  its  antigen.  This  is  determined  by  a  process  of  titration.  If,  for 
example,  an  antihuman  serum  is  to  be  titrated,  the  method  of  procedure 
is  as  follows : 

Secure  0.1  c.c.  of  fresh  human  serum  and  dilute  1  : 1000  by  adding 
99.9  c.c.  of  normal  saline  solution.  Of  this  dilution,  0.1  c.c.  is  equivalent 
to  the  standard  dose  of  0.0001  c.c.  of  undiluted  serum.  The  serum  may 
be  used  1  :  500.  The  antiserum  is  heated  to  55°  C.  for  half  an  hour  and 
diluted  1  :  10  (1  c.c.  immune  serum  plus  9  c.c.  of  saline  solution).  De- 
creasing doses  of  immune  serum  are  mixed  with  a  constant  dose  of  anti- 
gen and  complement.  At  the  same  time  the  anticomplementary  ti- 
tration of  the  immune  serum  is  made  by  substituting  salt  solution  for 
antigen.  The  doses  to  employ  and  the  results  of  an  actual  titration 
are  shown  in  Table  19: 

Tube  16  is  the  hemolytic  system  control,  and  shows  complete  hemol- 
ysis;  tube  15  is  the  antigen  control,  and  shows  complete  hemolysis, 
as  the  quantity  of  serum  is  too  small  to  exert  an  anticomplementary 
influence;  tubes  11  to  14  are  the  tests  for  anticomplementary  action  of 
the  antiserum.  In  the  present  instance  the  serum  was  several  months 
old  and  the  maximum  dose  of  1  c.c.  (  =  0.1  c.c.  undiluted  serum)  was 
very  slightly  anticomplementary.  A  fresh  serum  is  practically  never 
anticomplementary  in  this  dosage,  but  these  tubes  should,  nevertheless, 
be  included  in  each  titration.  Tubes  1  to  10  include  the  antigenic  titra- 
tion, and  show  that  the  antiserum  is  perfectly  antigenic  in  dose  of 


PROTEIN    DIFFERENTIATION    BY    COMPLEMENT    FIXATION  529 

0.3  c.c.  of  this  dilution  (  =  0.03  c.c.  undiluted  serum).     In  performing 
the  main  test  double  this  quantity,  or  0.6  c.c.,  would  be  used. 

TABLE  19.— TITRATION  OF  AN  IMMUNE  SERUM 


SHEEP'S 

SERUM 

COM- 

0 

ANTI- 

COR- 

PUS™ 

TUBE 

ANTI- 
SERUM, 
1:10,  C.c. 

ANTIGEN, 
1:1000, 
C.c. 

PLE- 
MENT, 
1:20 

I 

SHEEP 
AMBO- 

CEPTOR, 

CLES, 

(2.5 
PER 

RESULTS  AFTER  INCU- 
BATION FOR  ONE  AND 
ONE-HALF  HOURS 

C.c. 

& 

UNITS 

CENT.), 

« 

C.c. 

1.  . 

1.0 

0.1 

1 

1 

2 

1 

Inhibition    of    hem- 

§ 

olysis 

2  

0.9 

0.1 

1 

£3 

2 

1 

Inhibition    of    hem- 

rt 

olysis 

3  

0.8 

0.1 

1 

2 

1 

Inhibition    of   hem- 

1 

olysis 

4  

0.7 

0.1 

1 

2 

1 

Inhibition    of    hem- 

A  . 

olysis 

5  

0.6 

0.1 

1 

>» 

2 

1 

Inhibition    of    hem- 

r+=>t-. 

olysis 

6  

0.5 

0.1 

1 

o  tt 

2 

1 

Inhibition    of    hem- 

02 

olysis 

7  

0.4 

0.1 

1 

<U 

2 

1 

Inhibition    of    hem- 

3 

olysis 

8  

0.3 

0.1 

1 

.    -V 

2 

1 

Inhibition    of    hem- 

c 

olysis 

9  

0.2 

0.1 

1 

o 

2 

1 

Partial  inhibition  of 

10  

0.1 

0.1 

1 

ed 

2 

1 

hemolysis 
Slight  inhibition  of 

cr 

hemolysis 

11  

1.0 

0 

1 

£ 

2 

1 

Very    slight    inhibi- 

12.. 
13  

0.8 
0.4 

0 
0 

1 
1 

| 

2 
2 

1 
1 

tion  of  hemolysis 
Complete  hemolysis 
Complete  hemolysis 

14 

0.2 

o 

1 

0 

1 

Complete  hemolysis 

15 

0 

0.1 

1 

d 

40 
0 

1 

Complete  hemolysis 

16  

0 

0 

1 

3 

w 

A 

2 

1 

Complete  hemolysis 

Each  antiserum  is  tested  in  a  similar  manner.  In  forensic  blood 
tests  an  antihuman  serum  is,  of  course,  employed  first;  if  this  is  negative 
and  it  is  desirable  to  determine  the  source  of  the  blood,  other  antiserums, 
so  that  the  ox,  horse,  dog,  etc.,  are  prepared,  titrated,  and  tested  with  a 
solution  of  the  blood-stain. 

The  Blood-stain. — It  is  first  necessary  to  ascertain  that  the  stain  is  a 
blood-stain;  this  is  done  by  performing  the  hemin  crystal  or  an  oxydase 
test  (p.  326)u  The  stain  is  then  extracted  in  normal  saline  solution,  as 
described  on  p.  327.  A  1  :  1000  dilution  is  made  approximately  by  so 
diluting  the  extract  that  it  just  gives  a  slight  opalescence  when  boiled 
with  a  few  drops  of  acetic  acid,  and  a  slight  foam  persists  after  shaking. 

Unless  it  is  perfectly  clear,  it  should  be  filtered. 
34 


530  THE   TECHNIC    OF   COMPLEMENT-FIXATION    REACTIONS 

The  Test. — Into  a  series  of  six  small  test-tubes  place  increasing  doses 
of  extract  of  the  blood-stain  (antigen),  as  follows:  0.1  c.c.,  0.2  c.c.,  0.4 
c.c.,  0.6  c.c.,  0.8  c.c.,  1  c.c.;  add  double  the  titrated  dose  of  antiserum 
and  1  c.c.  of  complement  (1  :  20),  with  sufficient  salt  solution  to  bring 
the  total  volume  in  each  tube  up  to  3  c.c. 

The  following  controls  are  included : 

1.  Antigen  control;    1.0  c.c.  of  the  blood  extract  plus  1  c.c.  of  diluted 
complement  and  salt  solution. 

2.  Antiserum  control:   double  the  titrated  dose  plus  1  c.c.  of  diluted 
complement  and  salt  solution. 

3.  Hemolytic  control;   at  this  time,  1  c.c.  of  diluted  complement  and 
salt  solution. 

4.  Corpuscle  control:   1  c.c.  of  corpuscle  suspension  and  salt  solution. 
The  tube  should  be  plugged  with  cotton. 

Shake  all  the  tubes  gently  and  incubate  for  an  hour  at  37°  C.  in  a 
water-bath  or,  preferably,  six  hours  or  overnight  in  a  refrigerator  at 
8°  C.  Add  2  units  of  hemolytic  amboceptor  and  1  c.c.  of  corpuscle 
suspension  to  each  tube  except  the  corpuscle  control.  Shake  gently 
and  reincubate  for  from  one  to  two  hours  in  a  water-bath,  depending 
upon  the  degree  of  hemolysis  present  in  the  controls. 

The  readings  are  made  at  once,  and  again  after  the  tubes  have  been 
allowed  to  settle  in  the  refrigerator  overnight.  Inhibition  of  hemolysis 
with  the  smallest  dose  of  blood  extract — 0.1  c.c.  ( =  approximately  0.0001 
c.c.  of  blood) — indicates  that  the  blood  extract  is  most  certainly  the 
antigen  for  the  antiserum  employed.  Even  with  the  maximum  dose  of 
extract — 1  c.c.  (  =  approximately  0.001  c.c.  of  blood) — inhibition  of 
hemolysis  serves  to  show  the  nature  of  the  blood.  With  an  antihuman 
serum,  for  instance,  a  similar  specific  reaction  would  be  possible  only 
with  the  bloods  of  the  higher  apes. 

In  making  blood  tests  for  medicolegal  purposes  the  antiserum 
should  not  only  be  standardized  with  a  definite  dilution  of  human  serum, 
but  the  whole  test  should  first  be  conducted  with  a  known  dried  human 
blood-stain,  and  it  must  be  borne  in  mind  that  extreme  accuracy  in  all 
manipulations  is  essential. 

In  Table  20  are  shown  the  method  and  the  results  of  an  actual  test, 
using  a  dried  human  blood-stain  and  the  same  antiserum  as  previously 
directed. 

I  prefer  this  complement-fixation  test  to  the  precipitin  reaction  in 
the  differentiation  of  proteins,  as  the  readings  are  sharper  and  more 
definite.  This  test  is  fully  as  reliable  as  the  precipitin  test,  and  there 
is  less  danger  of  group  reaction. 


PROTEIN    DIFFERENTIATION    BY    COMPLEMENT    FIXATION 
TABLE  20.— FORENSIC  BLOOD  TEST 


531 


TUBE 

EXTRACT 

OF 

BLOOD- 
STAIN, 
1:1000 
C.c. 

ANTI- 
SERUM 
1:10, 
C.c. 

COM- 
PLE- 
MENT, 
1:20, 
C.c. 

id  incubated 

ANTI- 
SHEEP 
AMBO- 

CEPTOR, 

UNITS 

COR- 
PUS- 
CLES 
(2.5 
PER 
CENT.), 
C.c. 

RESULTS  AFTER  ONE 
AND  ONE-HALF  HOURS' 
INCUBATION 

1. 

0  1 

06 

1 

3 

2 

1 

Marked    inhibition 

2  

0.2 

0.6 

1 

1 

2 

1 

of  hemolysis 
Marked    inhibition 

3  
4  
5 

0.4 
0.6 
08 

0.6 
0.6 
06 

1 
1 
1 

^3 

02 

11 

•»**M 

Ji 

2 
2 
2 

1 

1 
1 

of  hemolysis 
Complete  inhibition 
of  hemolysis 
Complete  inhibition 
of  hemolysis 
Complete  inhibition 

6  

7 

1.0 
10 

0.6 
0 

1 
1 

O 

<N 

3 

2 
2* 

1 
1 

of  hemolysis 
Complete  inhibition 
of  hemolysis 
Antigen         control  * 

8  
9  
10  

0 
0 
0 

0.6 
0 
0 

1 
1 

o 

.§ 

-^ 
g 

1 

0 

.S 
H3 

2 
2 

o 

1 
1 
1 

hemolysis 
Antiserum     control  : 
hemolysis 
Hemolytic     control: 
hemolysis 
Corpuscle      control  * 

CO 

no  hemolysis 

2.  Complement-Fixation  Method  for  the  Identification  of  Meats. — 

The  technic  is  essentially  similar  to  that  used  in  the  foregoing  test. 
Antiserums  are  prepared  by  immunizing  rabbits  with  the  serums  of 
various  animals,  as  the  ox,  horse,  dog,  cat,  or  any  other  animal  the 
presence  of  whose  flesh  is  to  be  identified  in  sausages,  bologna,  etc.  It 
is  not  necessary  to  immunize  with  an  extract  of  these  meats  themselves, 
as  the  blood  or  blood-serums  will  suffice.  The  technic  of  immunization 
is  the  same  as  that  employed  in  the  preparation  of  precipitin  serums. 
Each  antiserum  is  titrated  with  its  antigen,  as  previously  described, 
and  is  used  in  double  the  titrated  dose  in  conducting  the  main  test. 

An  extract  of  the  flesh  to  be  examined  is  prepared  as  described  on 
p.  333. 

The  test  is  then  conducted  in  exactly  the  same  manner  as  previously 
described. 

3.  Complement-Fixation  Method  for  the  Identification  of  Bacterial 
Antigens. — As  a  means  of  diagnosis,  this  test  has  very  limited  practical 
value.     It  aims  to  detect,  by  means  of  complement  fixation  with  a 
known  antiserum,  a  soluble  bacterial  antigen  in  the  blood-serum  of  a 
patient.     For  example,  in  typhoid  fever  the  patient's  serum  is  mixed 
with  a  potent  antityphoid  serum  in  the  presence  of  complement.     After 


532  THE   TECHNIC    OF   COMPLEMENT-FIXATION   REACTIONS 

incubating  for  an  hour  at  37°  C.,  amboceptor  and  corpuscles  are  added 
to  test  for  free  complement.  An  absence  of  hemolysis  indicates  that 
complement  has  been  fixed  by  the  antiserum  and  soluble  typhoid  antigen 
in  the  serum  of  the  patient.  As  a  rule,  and  for  purposes  of  diagnosis, 
this  order  of  procedure  is  reversed:  the  antigen  is  furnished  and  then 
sought  for  in  the  patient's  serum  (p.  498),  as  in  the  gonococcus  fixation 
test,  syphilis  reaction,  etc. 

An  immune  serum  is  prepared  by  immunizing  rabbits  with  increasing 
doses  of  an  emulsion  of  the  bacteria  which  we  wish  to  test  for  in  the 
patient's  serum. 

The  bacterial  extract  is  prepared  as  follows:  Make  cultures  of  the 
bacteria  on  slants  of  agar;  wash  off  a  sufficient  number  with  normal 
saline  solution  until  20  or  30  c.c.  of  a  heavy  emulsion  are  secured;  add 
0.4  per  cent,  of  phenol,  arid  shake  mechanically  with  glass  beads  for 
twenty-four  hours;  then  heat  to  60°  C.  for  an  hour,  and  either  centri- 
fuge thoroughly  or  filter  through  a  Berkefeld  filter.  The  clear  filtrate 
should  be  preserved  in  a  tightly  stoppered  bottle  in  an  ice-chest.  It  is 
well  to  titrate  this  extract  for  its  anticomplementary  dose.  As  a  rule, 
these  extracts  are  free  from  anticomplementary  action  until  relatively 
large  doses  are  employed. 

The  antiserum  is  heated  to  55°  C.  for  half  an  hour  and  titrated  with 
0.01  c.c.  of  the  bacterial  extract  (0.1  c.c.  of  a  1: 10  dilution)  in  10  doses, 
ranging  from  0.1  c.c.  to.  1.0  c.c.  Double  the  dose  giving  complete  fixa- 
tion of  complement  is  used  in  testing  for  the  bacterial  antigen  in  human 
serum. 

In  conducting  this  test  the  patient's  serum  is  heated  to  55°  C.  for 
half  an  hour,  and  decreasing  doses,  ranging  from  0.5  c.c.  to  0.01  c.c., 
are  placed  in  a  series  of  test-tubes  together  with  double  the  titrated  dose 
of  antiserum.  Complement  and  salt  solution  are  now  added,  and  after 
incubating  for  an  hour  at  37°  C.,  amboceptor  and  corpuscles  are  added 
and  the  tubes  reincubated.  The  general  technic  and  controls  are  the 
same  as  those  previously  described. 

The  test  has  some  value  in  special  research  work,  but  for  practical 
use  it  has  given  way  to  the  agglutination  reactions  and  complement- 
fixation  tests  for  the  detection  of  antibody  with  a  known  antigen. 


COMPLEMENT-FIXATION  TEST  IN  CANCER 

None  of  the  various  complement-fixation  methods  that  have  been 
advocated  from  time  to  time  in  the  diagnosis  of  cancer  have  proved  of 


COMPLEMENT-FIXATION   TEST   IN   CANCER  533 

practical  value.  More  recently  von  Dungern1  has  advocated  a  method 
that  yielded  90  per  cent,  of  positive  reactions  in  known  cases  of  cancer. 
Positive  reactions  have  also  occurred  in  tuberculosis  and  syphilis,  and 
the  reports  of  various  other  investigators  are  somewhat  contradictory. 
Whitman,  in  a  study  of  30  cases,  found  the  method  highly  satisfactory. 
Lucke2  has  found  the  test  of  no  value  at  all. 

1  Munch,  med.  Wochenschr.,  1912,  59,  65,  1098,  2854. 

2  Personal  communication. 


CHAPTER  XXV 
CYTOTOXINS 

IN  Chapter  XXIII  the  cytolysins  in  general  were  considered, 
especially  their  theoretic  structure  and  the  mechanism  of  their  action. 

It  will  be  remembered  that  the  general  name  "cytolysin"  is  applied 
to  an  amboceptor  or  antibody  of  the  third  order  of  receptors  that  is 
capable  of  preparing  its  antigen  for  the  disintegrative  or  lytic  action  of  a 
complement.  The  two  best  known  and  most  important  members  of 
this  group  of  antibodies  have  been  considered,  namely,  the  hemolysins 
and  the  bacteriolysins. 

Following  the  discovery  of  the  hemolysins  and  the  bacteriolysins 
and  of  the  mechanism  of  their  action,  it  was  not  long  before  similar 
studies  were  undertaken  with  other  cells,  with  the  result  that  attempts 
have  been  made  to  prepare  immune  cytolytic  serums  for  practically 
every  organ  of  the  body.  This  outcome  was  but  natural,  in  view  of  the 
enormous  theoretic  importance  of  specific  cytolysins,  not  only  from  the 
additional  light  that  may  be  thrown  upon  physiologic  and  pathologic 
processes  in  general,  but  also  from  the  standpoint  of  specific  therapeutics. 

Nomenclature. — While  actual  lysis  or  solution  of  erythrocytes  and 
bacteria  may  be  brought  about  by  antibodies  of  this  order,  yet  actual 
solution  is  not  apparent  with  most  other  body-cells,  although  a  distinct 
toxic  action  may  be  observed.  For  instance,  an  antispermatozoa 
serum  will  cause  these  cells  to  lose  their  motility,  but  does  not  actually 
dissolve  them.  Hence  the  name  cytotoxin  has  been  applied  to  these 
immune  serums.  This  is  probably  a  better  term  than  cytolysin;  but 
it  is  to  be  remembered  that,  so  far  as  is  now  known,  both  cytolysins  and 
cytotoxins  are  antibodies  that  possess  the  same  nature  and  structure, 
except  that  in  the  former  group  the  process  is  complete  and  ends  in 
actual  lysis  of  the  cell.  The  term  cytolysin  is,  therefore,  more  appro- 
priately applied  to  the  bacteriolysin  and  hemolysins;  whereas  the  term 
cytotoxin  is  reserved  for  those  immune  serums  that  injure  their  cells  without 
complete  lysis  (a  toxic  action),  such  as  nephrotoxin,  hepatotoxin,  etc. 
This  chapter  is  mainly  concerned  with  the  latter  group. 

Nature  and  General  Properties  of  Cytotoxins. — As  previously 
stated,  cytotoxins  are  amboceptors  or  antibodies  of  the  third  order,  and 

534 


METHODS   OF   STUDYING   CYTOTOXINS  535 

possess  the  same  general  properties  as  the  bacteriolysins  and  hemoly- 
sins,  except  that,  as  will  be  pointed  out  later,  they  do  not  possess  the 
same  specificity.  Without  the  presence  of  a  complement  they  are 
inactive.  They  are  thermostabile,  possess  the  same  general  affinity  for 
their  antigen,  and  may  be  removed  from  a  serum  by  saturation  with 
the  antigen  in  a  manner  similar  to  that  used  for  the  removal  of  a 
hemolysin. 

Preparation  of  Cytotoxins. — Cytotoxic  serums  are  prepared  by  im- 
munizing an  alien  animal  with  fine  suspensions  of  cells  of  the  particular 
organ  being  studied.  (See  p.  73.)  Every  effort  should  be  made  to 
remove  all  traces  of  blood,  and  to  secure  as  pure  an  emulsion  of  the  same 
cells  and  to  work  as  aseptically  as  possible.  Injections  are  best  given 
intraperitoneally,  rabbits  being  well  adapted  for  the  preparation  of 
these  serums. 

Beebe l  has  made  the  statement  that  more  specific  serums  are  obtained 
if  the  nucleoproteins  are  isolated  and  used  in  the  process  of  immuniza- 
tion, than  if  the  cells  themselves  are  used.  Wells,2  however,  believes 
that  the  nucleoproteins  of  cells  are  not  specific  in  character,  and  Pearce, 
Karsner,  and  Eisenbrey 3  found  that  nephrotoxic  and  hepatoxic  serums 
prepared  by  the  injection  of  the  nucleoproteins  of  these  cells,  were  no 
more  toxic  than  serums  prepared  from  the  globulins  and  albumins  of  the 
same  organs. 

Methods  of  Studying  Cytotoxins. — While  the  phenomena  of  hemolysis 
and  bacteriolysis  may  readily  be  observed  in  experiments  in  vitro,  the 
influence  of  other  cytotoxins  on  the  particular  cells  used  as  antigens  is 
more  difficult  to  determine.  The  methods  of  exposing  cell  emulsions 
to  the  action  of  the  serum  have  not  been  found  satisfactory  for  testing 
the  specificity  of  cytotoxins. 

The  technic  generally  employed  consists  in  making  subcutaneous, 
intraperitoneal,  or  intravenous  injections  of  the  immune  serum  into  the 
animal,  or  into  the  arteries  leading  to  particular  organs.  Functional  dis- 
turbances and  delicate  histologic  changes  in  various  organs  have  served  as 
criteria  for  determining  the  degree  of  specificity  that  exists.  The  loss  of 
some  manifestation  of  vitality  on  the  part  of  the  cell,  as  a  loss  of  motility 
(spermatozoa)  or  an  inability  to  proliferate,  may  aid  in  studying  the 
effect  of  these  serums. 

More  recently  several  other  methods  have  been  used,  especially  the 

1  Jour.  Exper.  Med.,  1905,  vii,  733. 

2  Chemical  Pathology,  1914,  Second  Edition,  W.  B.  Saunders  Co. 

3  Jour.  Exper.  Med.,  1911,  xiv,  44. 


536  CYTOTOXINS 

complement-fixation  and  the  epiphanin  reactions  and  that  of  observing 
the  influence  of  cytotoxic  serums  upon  cells  grown  in  vitro  (Lambert). 

Specificity  of  Cytotoxins. — As  has  been  stated  elsewhere,  the  hemoly- 
sins  and  the  bacteriolysins  are  highly  specific,  especially  the  former 
group.  With  the  cytotoxins,  however,  this  specificity  is  not  observed. 
Most  cytotoxic  serums  are  also  hemolytic,  notwithstanding  the  fact  that 
careful  precautions  have  been  taken  to  remove,  so  far  as  possible,  all 
traces  of  blood  from  the  inoculum  during  the  process  of  immunization. 
Metchnikoff  found  a  spermatotoxic  serum  to  be  also  hemolytic,  but  he 
believed  that  this  property  could  be  removed  by  treating  the  immune 
serum  with  the  corresponding  corpuscles,  and  in  this  manner  dissolve 
out  the  hemolysin.  Numerous  other  investigators  have  found,  however, 
that  cytotoxic  serums  may  attack  the  cells  of  other  organs,  as  well  as 
those  that  have  been  used  as  their  antigens. 

The  subject  has  been  very  carefully  investigated  by  Pearce.1  The 
injection  of  an  antidog  nephrotoxic  serum  prepared  by  immunizing 
rabbits  with  washed  dog  kidney  is  followed  by  the  development  of  a 
tubular  nephritis,  with  albuminuria  and  occasionally  hemoglobinuria, 
and  accompanied  by  granular  degeneration  of  the  liver.  These  serums 
are  usually  hemolytic  in  vitro.  Similarly,  in  a  study  of  hepatotoxic 
serums,  Pearce  found  that  the  most  striking  lesions  were  referable  to 
the  hemagglutinating  and  hemolytic  properties  of  the  serum,  causing 
thrombosis,  embolism,  and  hemorrhages,  whereas  secondary  necroses 
may  be  caused  by  a  direct  toxic  action  of  the  serum  on  certain  parenchy- 
matous  cells. 

Pearce,  Karsner,  and  Eisenbrey  found  that  the  serums  of  rabbits 
injected  repeatedly  with  the  nucleoproteins,  globulins,  and  albumins  of 
the  liver  and  kidney  of  the  dog,  gave  no  evidence  of  organ  specificity 
in  vitro  or  in  vivo  experiments.  These  investigators  were  not  able  to 
support  the  view  put  forward  that  nucleoproteins  play  an  important 
part  in  the  production  of  cytotoxic  immune  serums. 

Lambert2  has  recently  studied  the  subject  with  cultures  of  rat  sar- 
coma and  rat  embryo  skin  and  their  immune  serums,  and  found  that 
these  cytotoxins  were  not  specific  for  the  tissue  injected. 

These  results  are  not  surprising  when  it  is  remembered  that  all  the 
body-cells  have  a  common  origin,  and  that,  although  the  cells  of  various 
organs  may  differ  considerably  in  morphologic  and  functional  characters, 
they  have  certain  receptors  in  common,  and,  as  Pearce  originally  main- 

1  Jour.  Exper.  Med.,  1914,  xix,  277. 

2  Univ.  Penna.  Med.  Bull.,  1903,  xvi,  217;  Jour.  Med.  Research,  1904,  xii,  1 


AUTOCYTOTOXINS  537 

tained,  it  is  hardly  conceivable  that  specific  somatogenic  cytotoxins 
can  be  produced. 

Autocytotoxins. — While  these  toxins  possess  some  theoretic  interest, 
they  are  of  very  rare  occurrence  in  experimental  work.  Certainly  cells 
of  the  kidney,  liver,  and  other  organs  are  constantly  dying  and  being 
replaced  by  new  cells;  the  receptors  of  these  cells  are  thereby  set  free, 
and  are  capable  of  forming  a  union  with  the  receptors  of  other  cells,  and 
the  possibility  for  the  formation  of  autocybotoxins  is  established.  Ac- 
cording to  Ehrlich,  however,  the  side-arms  anchoring  the  receptors  of 
the  dead  cells  are  sessile  in  nature,  and  are  unlikely  to  cause  overproduc- 
tion, as  in  the  case  of  antibodies  for  bacterial  substances  or  for  the  cells 
of  other  species.  On  the  other  hand,  a  simultaneous  production  of 
antiautotoxins  that  counteract  the  autotoxins  and  preserve  a  delicate 
physiologic  equilibrium  may  occur,  the  whole  subject  being,  however, 
still  in  the  experimental  stage. 

Of  most  interest  in  this  connection  are  the  theoretic  autonephrotoxins. 
These  may  be  produced  when  part  of  a  kidney  becomes  disorganized 
in  the  living  body,  as  by  means  of  a  toxin.  Theoretic  autotoxins  may 
then  be  produced,  which,  acting  upon  other  kidney  cells,  institute  a 
vicious  cycle.  Acting  upon  these  assumptions,  Ascoli  and  Figari 1  and 
Lindeman  2  have  proposed  a  new  theory  as  to  the  pathogenesis  of  certain 
of  the  nephritides.  These  observers  would  account  for  the  cardiac 
hypertrophy  of  nephritis  by  attributing  it  to  the  action  of  the  nephro- 
toxic  serum  in  causing  contraction  of  the  peripheral  vessels,  with  con- 
sequent increase  of  blood-pressure;  the  nephritic  nervous  symptoms, 
they  believe,  are  due  to  the  fact  that  the  serum  contains  a  neurotoxic 
constituent. 

Lindeman  has  produced  a  toxic  nephritis  in  dogs  by  giving  them 
injections  of  potassium  bichromate,  and  found  that  the  serum,  although 
free  from  the  chromate,  was  toxic  to  other  dogs,  a  finding  he  believed 
due  to  the  presence  of  antonephrotoxins  produced  in  the  first  dog  as  a 
result  of  the  destruction  of  kidney  cells.  According  to  this  view,  the 
original  toxic  cause  of  a  degenerative  nephritis  would  be  less  responsible 
for  the  continuance  of  the  process  than  would  the  formation  of  an 
autonephrotoxin.  While  these  conclusions  are  somewhat  far-reaching, 
they  serve  to  indicate  that  the  same  processes  operative  in  bacterial 
infection  and  immunity  may  have  an  important  relation  to  other  patho- 
logic conditions. 

1  Berl.  klin.  Wochenschr.,  1902,  xxxix,  634. 

2  Ann.  de  1'Inst.  Pasteur,  1900,  xiv,  49. 


538  CYTOTOXINS 

It  is  not  rarely  observed  that  in  large  tumors  and  similar  lesions 
certain  groups  of  cells  may  undergo  digestion,  but  in  these  the  lysis  is 
commonly  ascribed  to  the  action  of  ferments  liberated  upon  the  death 
of  cells. 

Isocytotoxins  have  been  produced  experimentally,  as,  for  example, 
by  Ehrlich,  who  produced  isohemolysins  by  injecting  goats  with  goat 
blood,  and  by  Metchnikoff,  who  prepared  isospermatoxic  serums. 

Anticytotoxic  serums  have  likewise  been  prepared  by  careful  im- 
munization with  cytotoxic  serums. 

Varieties  of  Cytotoxins. — As  previously  stated,  attempts  have  been 
made  to  prepare  cytotoxic  serums  for  practically  all  the  organs  and  tis- 
sues. Since  none  of  these  has  been  found  to  be  absolutely  specific, 
and  hence  since  they  possess  little  or  no  practical  value,  they  will  receive 
but  brief  consideration  here. 

1.  Spermatotoxin. — This   serum   was   prepared   simultaneously   by 
Metchnikoff1  and  Landsteiner  in  1899,  and  was  one  of  the  earliest 
cytotoxins  to  be  studied.     It  is  a  hemolytic  serum,  and  causes  sperma- 
tozoa to  lose  their  motility.     It  would  seem  to  affect  also  the  vitality 
of  the  spermatozoa  in  vivo,  inasmuch  as  De  Lester,  by  the  injection  of 
this  serum,  rendered  male  mice  sterile  for  from  sixteen  to  twenty  days. 

2.  Epitheliotoxin. — A  cytotoxic  serum  for  the  ciliated  epithelium  of 
the  trachea  was. prepared  by  von  Dungern.2    The  cells  became  dis- 
integrated in  the  peritoneal  cavity  of  the  immunized  animal,  but  not  in 
that  of  the  normal  animal.     This  serum  also  proved  to  be  hemolytic. 

Similar  serums  have  been  prepared  with  cancer  cells,  in  the  hope 
of  establishing  a  specific  serum  therapy,  but  all  efforts  have  thus  far 
proved  futile. 

3.  Leukotoxins. — This  serum  was  first  prepared   by  Metchnikoff3 
and  Besredka4  by  injecting  the  spleen  of  rats  into  guinea-pigs.     The 
serums  have  also  been  prepared  by  effecting  immunization  with  exudates 
rich  in  leukocytes  or  with  the  emulsion  of  lymphoid  organs  (Flexner 
and  Ricketts) .     They  are  usually  hemolytic,  and  also  attack  endothelial 
cells.     Their  action  may  be  observed  in  vitro  when  the  leukocytes  lose 
their  ameboid  motility  and  the  protoplasm  swells,  clears,  and  may  dis- 
integrate, leaving  the  nucleus.  ..,..'- 

4.  Nephrotoxin. — This  serum  is  best  adapted  for  experimental  studies 

1  Ann.  de  PInst.  Pasteur,  1900,  xiv,  369. 

2  Munch,  med.  Wochenschr.,  1899,  xlvi,  1228. 

3  Ann.  de  1'Inst,  Pasteur,  1899,  xiii,  737. 

4  Ann.  de  Tlnst.  Pasteur,  1900,  xiv,  402. 


VARIETIES    OF   CYTOTOXINS  539 

of  the  cytotoxins.  As  shown  by  Pearce,  with  the  aid  of  this  serum 
physiologic  and  anatomic  alterations  and  lesions  are  readily  studied. 
The  injection  of  a  nephrotoxic  serum  produces  a  tubular  nephritis,  with 
albuminuria  and  possibly  hemoglobinuria.  The  serums  are  usually 
hemolytic,  and  frequently  cause  degenerative  lesions  in  the  liver,  due  in 
part  to  hemolysis  and  hemagglutination  of  red  corpuscles. 

5.  Hepatotoxin. — Delezenne l  was  the  first  to  work  with  the  so-called 
hepatotoxin,  which,  he  claimed,  possessed  absolute  organ  specificity. 
Subsequent  investigations,  however,  have  brought  forth  contradictory 
findings.     Pearce  found  that  hyaline  thrombi,  formed  of  agglutinated 
red  corpuscles,  are  primarily  responsible  for  the  areas  of  necrosis  and 
hemorrhage,  with  secondary  effects,  which  may  be  ascribed  to  a  cytotoxin 
liberated  chiefly  by  the  cells  in  the  thrombus,  and  acting  on  the  liver- 
cells.     These  findings  and  views  have  recently  received  support  from 
the  investigations  of  Karsner  and  Aub.2 

6.  Gastrotoxin. — Gastrotoxic  serums  have  been  studied  by  Bolton,3 
who  immunized  rabbits  with  emulsions  of  the  mucosa  of  the  stomach 
of  the  guinea-pig.     The  injection  of  this  serum  into  guinea-pigs  was 
followed  by  the  development  of  areas  of  hemorrhage,  necrosis,  and  ulcer 
formation  that  resembled  peptic  ulcers.     According  to  Bolton,  if  the 
gastric  secretions  were  neutralized  with  large  quantities  of  alkali,  the 
ulcers  did  not  develop,  indicating  that  the  peptic  ferments  may  be  oper- 
ative in  the  digestion  of  the  cells  after  their  destruction  by  the  immune 
serum.     Gastrotoxic  serums  were  found  to  produce  precipitates  with 
clear  filtrates  of  gastric  cells,  and  were  also  shown  to  be  hemolytic. 

7.  Synocytotoxin. — This  serum  has  been  produced  experimentally 
by  immunization  with  an  emulsion  of  placental  cells.     According  to 
Liepmann,4  it  produces  a  precipitate  with  a  filtrate  of  placenta  cells,  and 
at  one  time  it  was  believed  that  it  might  constitute  a  diagnostic  test  for 
pregnancy. 

Syncytotoxins  are  interesting  as  considered  in  reference  to  eclampsia 
and  other  toxemias  of  pregnancy.  As  is  well  known,  placental  cells 
may  become  detached  and,  gaining  entrance  to  the  circulation,  become 
lodged  in  remote  organs  (Schmorl).  This  has  given  rise  to  the  theory 
that  a  placentotoxin  is  developed  that  produces  the  nephritis  of  preg- 
nancy and  necrotic  lesions  in  the  liver.  Weichardt  asserts  that,  by 
digesting  placenta  in  vitro  with  an  active  placentotoxic  serum  and  in- 

1  Semaine  Med.,  1900,  xx,  290.  2  Jour.  Med.  Research,  1913,  xxviii,  377. 

3  Proc.  Roy.  Soc.,  Ixxvii,  426,  and  Ixxix,  533. 

4  Deutsch.  med.  Wochenschr.,  1902,  xxviii,  911. 


540  CYTOTOXINS 

jecting  the  digestate,  he  produced  symptoms  resembling  eclampsia  in  the 
lower  animals.  It  was  hoped  that  an  anticytotoxic  serum  might  be  pre- 
pared to  combat  the  effects  of  the  placentotoxin,  but  this  hope  has  not 
been  realized.  Renewed  interest  in  this  particular  subject  has 
been  manifested  by  the  recent  studies  of  Abderhalden  in  ferments  as 
applied  to  the  diagnosis  of  pregnancy  (p.  258). 

8.  Neurotoxin. — This  toxin  has  been  prepared  and  studied  by  Dele- 
zenne,  Centanni,  Delille,  and  others,  by  immunization  experiments  with 
emulsions  of  cerebrum,  cerebellum,  and  spinal  cord.     When  injected 
into  the  brain  direct,  these  serums  may  cause  profound  intoxication  of 
the  nerve-centers,  with  torpor  or  convulsions,  subnormal  temperature, 
and  death.     When  injected  directly  into  the  veins,  they  are  usually 
without  effect.     In  addition  to  their  neurotoxic  action,  they  are  generally 
hemolytic,  and  frequently  endotheliotoxic  and  leukotoxic. 

9.  Thyrotoxins. — This  serum  is  prepared  by  immunizing  animals 
with  emulsions  of  thyroid  gland.     Thyrotoxins  were  quite  prominently 
before  the  profession  a  few  years  ago,  owing  to  the  work  of  Beebe,  who 
advocated  their  use  in  the  treatment  of  various  goiters.     They  have  not 
fulfilled  their  expectations,  however,  since  they  may  also  produce  de- 
generative changes  in  the  various  organs,  as  the  liver,  spleen,  and  kid- 
neys. 

ROLE  OF  CYTOTOXINS  IN  IMMUNITY 

It  is  apparent  that,  according  to  our  present  knowledge,  the  cyto- 
toxins  proper,  although  they  possess  great  theoretic  importance  from 
their  possible  relationship  to  the  removal  and  disposal  of  enfeebled  and 
dead  cells,  occupy  a  subsidiary  place  in  the  processes  of  immunity.  The 
processes  governing  these  changes  are  finally  and  delicately  balanced, 
and  although  obscure,  they  offer  an  intricate  but  fascinating  field  for 
research. 

If  the  ferments  concerned  in  Abderhalden's  studies  are  related  in 
any  way  to  the  cytolysins,  the  subject  becomes  of  great  interest,  and  a 
new  field,  with  immense  possibilities,  is  opened  for  further  study. 

Practical  Applications. — (1)  In  therapeutics  the  cytotoxins  have  not 
established  a  place  for  themselves  and  their  use  has  been  disappointing. 
As  previously  stated,  the  use  of  thyrotoxic  serums  has  not  met  with 
considerable  success;  epitheliotoxic  serums  have  likewise  not  been 
efficient  in  the  treatment  of  cancer.  They  possess  theoretic  interest, 
however,  from  the  possibility  of  their  so  injuring  glands  that  their  func- 
tions may  be  studied;  theoretically,  the  use  of  minute  and  carefully 


CYTOTOXIC    REACTIONS  541 

graded  doses  of  hemolytic  serums  may  effect  the  production  of  anti- 
hemolysins  and  aid  in  the  treatment  of  certain  anemias. 

(2)  In  diagnosis  cytotoxic  reactions  have  been  employed  by  Freund 
and  Kaminer.  These  observers  used  the  cytotoxins  as  a  diagnostic 
aid  in  cancer,  but  with  indifferent  success.  Abderhalden's  pregnancy 
test  possesses  some  practical  value,  and  a  similar  technic  has  been  suc- 
cessfully employed  in  the  diagnosis  of  cancer.  The  work  of  Abderhalden 
has  served  to  open  up  a  comparatively  new  and  intensely  interesting 
field,  which,  when  fully  developed,  as  the  result  of  overcoming  difficult 
technical  procedures,  may  offer  additional  means  in  the  diagnosis  of 
various  diseases.  This  test  has  been  described  elsewhere  under  the  head 
of  Ferments  (p.  265). 

CYTOTOXIC  REACTIONS 

Cytolytic  Cancer  Diagnosis  of  Freund  and  Kaminer.1 — This  reaction 
is  based  upon  the  observation  that  while  normal  serum  has  the  power 
to  dissolve  cancer  cells,  the  serum  of  cancerous  persons  lacks  this  prop- 
erty, and  has  the  power  to  inhibit  the  destruction  of  such  cells  by  normal 
serum. 

The  same  authors  have  also  observed  that  when  cancer  serum  is 
mixed  with  an  extract  of  cancer  cells  a  precipitate  forms.  They  claim 
to  have  secured  88  per  cent,  of  positive  reactions  in  113  cases  exam- 
ined, and  believe  that  the  reaction  occurs  early  enough  and  is  suf- 
ficiently specific  to  render  it  of  practical  value.  These  observations, 
however,  have  not  been  sufficiently  confirmed. 

An  emulsion  of  cancer  cells  is  prepared  by  grinding  the  undegenerated 
portions  of  a  tumor,  freed  as  much  as  possible  from  fat  and  fibrous 
tissue,  in  a  mortar  and  adding  about  five  volumes  of  1  per  cent,  sodium 
biphosphate.  The  suspension  is  filtered  through  several  layers  of  gauze, 
and  after  the  cells  have  become  precipitated,  the  supernatant  fluid  is 
decanted.  The  residue  of  cells  is  washed  with  0.6  per  cent,  sodium 
chlorid  and  allowed  to  settle  again,  the  supernatant  fluid  is  decanted, 
and  the  residue  covered  with  1  per  cent,  sodium  fluorid.  The  last- 
named  fluid  must  first  be  neutralized  against  alizarin  until  only  a  trace 
of  the  violet  color  remains.  The  emulsion  will  keep  for  several  weeks 
in  an  ice-chest. 

Serum.— The  patient's  serum  should  be  collected  just  a  few  hours 
(not  over  twenty-four)  before  the  test  is  to  be  made,  and  must  be  clear 
and  free  from  cellular  elements. 

1  Biochem.  Zeitschr.,  1910,  26,  312;  Wien.  klin.  Wochenschr.,  1910,  23,  378,  and 
1221;  ibid.,  1911,  24,  1759. 


542  CYTOTOXINS 

The  Test. — To  10  drops  of  the  patient's  serum  add  one  drop  of  0.5 
per  cent,  solution  of  sodium  fluorid.  Then  add  one  drop  of  the  cancer- 
cell  emulsion  so  diluted  that  when  one  drop  of  the  mixture  is  placed  in  a 
blood-counting  chamber,  about  10  to  20  cancer  cells  will  be  found  in  a 
field  of  four  large  squares.  Close  the  counting  chamber  carefully,  ring 
with  vaselin  to  prevent  evaporation,  and  place  in  the  incubator  for 
twenty-four  hours. 

A  second  slide  is  prepared  from  a  mixture  composed  of  one  volume 
each  of  normal  serum,  cancer  serum,  and  0.6  per  cent,  sodium  chlorid 
and  sufficient  cell  emulsion. 

A  third  slide  is  prepared  with  a  fresh  normal  serum  in  the  same 
manner  as  when  the  patient's  serum  is  used. 

All  slides  are  incubated  for  twenty-four  hours  at  37°  C.  and  the  cells 
counted.  A  material  reduction  in  the  number  of  cells  with  the  normal 
serum  will  be  noted;  if  the  patient  has  carcinoma,  the  first  and  second 
slides  will  not  show  this  reduction,  whereas  if  the  patient  is  free  from 
cancer,  similar  reduc.tions  will  be  found  in  all  three  slides. 

The  authors  recommend  that  both  the  cytolytic  and  the  precipitin 
test  be  conducted  when  enough  serum  is  available  for  both. 


CHAPTER  XXVI 
THE  RELATION  OF  COLLOIDS  AND  LIPOIDS  TO  IMMUNITY 

WHILE  at  the  present  time  Ehrlich's  side-chain  theory  best  explains 
the  specificity  and  mode  of  action  of  various  antibodies,  there  is  a  grow- 
ing tendency  to  explain  many  of  these  reactions  on  a  physicochemical 
and  colloidal  basis. 

From  the  fact  that,  without  exception,  antigens  are  colloids,  and  that 
antibodies  also  are  colloid  in  their  chemical  characters,  it  is  advisable 
to  review  briefly  some  of  the  main  facts  and  theories  concerning  these 
bodies  and  their  reactions. 

Varieties  of  Colloids. — Colloids  may  be  composed  of  two  different 
classes  of  substances: 

1.  Organic  substances,  as,  e.  g.,  all  forms  of  proteins  and  also  gums, 
starch,  glycogen,  tannin,  chondrin,  the  greater  number  of  organic  dyes, 
and  probably  the  enzymes. 

2.  Inorganic  substances,  as,  for  example,  the  inorganic  colloids,  such 
as  silicic  acid,  ferric  hydroxid,  arsenic  sulphid,  and  many  other  similar 
compounds. 

Since  the  living  tissues  and  fluids  are,  without  exception,  colloids  and 
colloidal  solutions,  the  properties  of  the  cells  are  largely  the  properties  of 
colloids. 

Nature  and  Properties  of  Colloids. — Since  Graham,1  in  1861,  studied 
the  differences  between  the  substances  that  did  or  did  not  diffuse  readily 
through  animal  or  parchment  membranes,  soluble  substances  have  been 
classified  in  two  main  groups :  (a)  Colloids,  or  those  substances  that  were 
dissolved  to  the  extent  of  showing  no  visible  particles  in  suspension,  but 
that  did  not  pass  through  diffusion  membranes  at  all,  or  did  so  very 
slowly  indeed,  and  (6)  crystalloids,  or  solutions  that  diffuse  through 
membranes  quite  readily. 

Since  Graham's  discoveries  investigations  have  shown  that  any  and 
all  substances  may  be  either  colloid  or  crystalloid,  depending  upon  the 
treatment  they  receive;  thus  albumin  may  be  crystallized  and  common 
table  salt  obtained  in  a  state  of  colloidal  solution.2  Furthermore,  there 
is  much  evidence  indicating  that  all  colloid  systems  are  unstable  and 

1  Phila.  Trans.,  1861,  183. 

2  V.  Weimarn,  Ztsch.  f.  Chem.  Ind.  Koll.,  1908,  3,  26;  ibid.,  9110,  7,  92. 

543 


544         THE    RELATION    OF    COLLOIDS   AND    LIPOIDS   TO    IMMUNITY 

never  in  equilibrium;  conditions  which  determine  the  appearance  of  a 
body  in  the  colloid  or  crystalline  form  appear  to  indicate  that  bodies 
always  separate  from  solution  in  the  amorphous  or  colloidal  condition 
and  that  all  crystallization  is  a  secondary  phenomenon. 

On  the  other  hand,  we  may  have  substances  that  are  quite  insoluble 
when  aggregated  in  masses,  but  when  derived  in  pure  form  by  me- 
chanical means  can  be  suspended  and  uniformly  distributed  through  a 
fluid  without  showing  any  marked  tendencjr  to  precipitate.  Such 
suspensions  or  emulsions  contain  particles  that  are  visible  under  the 
microscope;  they  usually  appear  turbid,  do  not  transmit  electricity,  and 
are  not  diffusible.  Colloids  occupy  a  place  between  the  true  solutions  of 
crystalloids  and  the  emulsions.  Sharp  boundaries  cannot  usually  be 
drawn  between  any  of  the  members  of  the  series.  They  differ  quanti- 
tatively in  some  manner  from  the  true  solutions  and  the  emulsions,  but 
may  approach  them  closely,  and  sometimes  resemble  them  so  strongly 
as  to  be  almost  indistinguishable  from  them.  For  the  most  part, 
however,  they  show  decided  characteristics  that  will  differentiate  them 
from  the  crystalloids,  on  the  one  hand,  and  the  suspensions,  on  the  other. 

Those  colloids  that  closely  resemble  the  true  solution  have  been 
designated  "  colloidal  solutions/'  and  those  resembling  more  closely  the 
suspensions,  "colloidal  suspensions."  Of  the  two  types,  the  colloidal 
solutions  are  far  more  important  biologically,  since  the  colloidal  suspen- 
sions are  usually  prepared  artificially  and  seldom  occur  in  nature. 

Colloids,  therefore,  appear  to  be  suspensions  of  masses  of  molecules, 
or  perhaps  of  very  large  single  molecules.  When  these  aggregations 
are  sufficiently  large,  we  have  an  ordinary  suspension. 

When  colloids  occur  in  a  highly  dispersed  state  they  are  called  sols; 
when  in  an  undispersed  or  but  slightly  dispersed  state  they  are  spoken 
of  as  gels.  A  colloid  substance  may  be  converted  from  a  sol  state  into 
a  gel  state  and  back  again,  when  it  is  called  a  reversible  colloid  or  emulsion; 
a  colloid  which  refuses  to  redisperse  is  an  irreversible  colloid  or  sus- 
pension. 

1.  Colloids  are  usually  amorphous  in  character,  and  with  few  excep- 
tions do  not  present  a  typical  structure;  they  are  not  crystalline  under 
any  visible  condition.     This,  however,  is  not  invariably  the  case,  for 
we  may  have  a  'protein,  like  hemoglobin,  which  resembles  a  typical  col- 
loid in  every  respect,  and  may  yet  form  crystals  readily  and  abundantly. 

2.  Colloids  do  not  form  true  solutions,  but  the  solvent  is  probably  an 
important  factor  in  determining  whether  or  not  a  substance  is  colloidal 
in  nature;   e.  g.,  soaps  form  true  solutions  in  alcohol  and  colloidal  solu- 


NATURE    AND    PROPERTIES    OF    COLLOIDS  545 

tions  in  water;  rubber  forms  colloidal  solutions  in  ether,  but  not  in 
water.  The  term  colloidal  solution  does  not,  therefore,  refer  to  a  true 
solution  in  the  sense  of  a  crystalloid,  but  to  a  colloidal  state  of  suspen- 
sion (the  so-called  colloidal  solution). 

3.  Colloids  are  non-diffusible,  or  lack  the  power  of  passing  through 
animal  and  parchment  membranes.      Not  all  colloids  possess  the  same 
rate  of  diffusion,  this  property  being  relative,  rather  than  absolute; 
however,  solutions  of  salts  (crystalloids)  pass  through  so  readily  that 
they  are  easily  separated  from  proteins  (colloids)  by  dialyzation,  a 
process  that  is  in  constant  practical  use. 

4.  Colloids  have  an  extremely  small  osomotic  pressure.    They  may, 
to  a  very  slight  degree,  exert  some  influence  upon  osmotic  pressure,  the 
freezing  and  boiling-points  of  fluids,  but  in  all  cellular  processes  in  which 
manifestations  of  osmotic  pressure  or  diffusion  are  present  the  crystal- 
loids may  be  considered  as  almost  entirely  responsible  for  these. 

5.  The  colloids  exhibit  surface  tension  to  a  high  degree — in  other  words, 
colloid  fluids  possess  the  force  that  strives  to  reduce  its  free  surface  to  a 
minimum.     As  partial  expressions  of  this  force,  the  formation  of  emul- 
sions when  oil  and  water  are  mixed  and  the  ameboid  movements  of  the 
ameba  and  leukocytes  may  be  mentioned  as  examples. 

6.  Colloids  do  not  separate  freely  into  ions  when  dissolved,  and  accord- 
ingly do  not  conduct  electricity  to  an  appreciable  extent.     When  an  electric 
current  is  passed  through  a  colloidal  fluid,  most  of  the  colloids  move 
toward  the  anode;    this  phenomenon,  known  as  cataphoresis,  is  also 
generally  exhibited  by  suspensions,  and  in  this  particular  the  colloids 
resemble  suspensions. 

7.  Colloids  are  usually  easily  precipitable  and  coagulable,  and  this  is 
readily  understood  when  the  slender  margin  that  exists  between  many  of 
the  colloids  and  the  suspensions  is  borne  in  mind.     Relatively  slight 
changes,  such  as  exposure,  gentle  heat,  the  presence  of  large  quantities 
of  crystalloids,  the  action  of  enzymes,  etc.,  may  throw  an  organic  colloid 
out  of  solution,  and  when  once  precipitated,  it  is  often  incapable  of  again 
dissolving  in  the  same  solvent.     Colloids  are  also  precipitated  by  many 
electrolytes,  apparently  through  the  formation  of  true  ion  compounds. 

8.  The  physical  structure  and  size  of  colloids.    This  subject  has  been 
studied  extensively  by  Hardy.1    Cells  contain  but  one,  type  of  colloids, 
the  proteins  that  form  non-reversible  coagula.    So  long  as  a  colloid  is  in 

1  A  good  general  outline  of  the  subject  of  colloids  may  be  found  in  Pauli's  "  Phys- 
ical Chemistry  in  the  Service  of  Medicine,"  1907,  translated  by  Fischer  (Chapman 
and  Hall). 

35 


546          THE    RELATION    OF    COLLOIDS   AND    LIPOIDS    TO    IMMUNITY 

solution  it  is  structureless;  but  such  solutions  may  become  solid  as  the 
result  of  changes  of  temperature  and  other  physical  means  and  from 
admixture  with  certain  chemical  fixing  agents.  The  structure  of  the 
coagula  varies  according  to  the  concentration  of  the  colloidal  solution 
and  the  nature  of  the  coagulant,  but  in  general  the  figures  obtained  in 
the  solidification  of  protein  solutions  by  such  fixing  agents  as  mercury 
bichlorid  and  formalin  bear  a  striking  resemblance  to  the  finer  structure 
of  protoplasm  as  described  by  cytologists.  These  facts,  no  doubt,  have 
an  important  bearing  upon  the  various  "foam,"  "reticular,"  and 
"pseudo-alveolar"  structures  of  the  protoplasm  of  cells  described  by 
Biitschli,  Fromann,  Arnold,  Reinke,  and  others,  and  may  indicate  the 
effect  of  fixatives  upon  colloid  solutions,  explaining  the  usual  time-worn 
objections  to  theories  of  protoplasmic  structure  as  based  upon  arti- 
ficial conditions  not  present  in  the  normal  living  cell,  and  variously 
interpreted  according  to  the  fixative  employed. 

Studies  in  the  size  of  colloidal  particles  have  been  greatly  facilitated 
by  dark  field  illumination  of  the  microscopic  field  or  the  so-called  ul- 
tramicroscope  devised  by  Siedentopf  and  Zsigmondy.  Various  other 
methods  have  been  devised  for  studying  the  size  of  colloidal  particles, 
as  ultrafiltration  by  Bechhod1;  the  weight  of  a  dispersed  substance  in  a 
given  volume  by  chemical  or  other  analysis;  by  studies  in  the  density 
of  the  dispersed  substance  and  other  methods.  These  studies  have 
indicated  that  colloidal  particles  are  usually  round  or  at  times  ovoid,  as 
indicating  beginning  crystallization;  that  the  main  difference  between 
suspensions  and  colloidal  solutions  is  one  of  dispersion  and  not  relative 
size  of  particles,  and  that  in  a  true  colloidal  solution  particles  of  widely 
differing  sizes  may  be  found  side  by  side. 

9.  Colloids  may  be  precipitated  by  electrolytes  of  opposite  sign,  as  well 
as  by  colloids.  In  a  colloidal  solution  surface  tension  constantly  tends 
to  make  the  particles  of  colloid  approach  one  another,  so  that  the  surface 
may  become  as  small  as  possible,  and  in  this  manner  brings  about  pre- 
cipitation or  coagulation. 

In  a  stable  solution  this  action  is  counterbalanced  by  a  force  of 
electric  repulsion.  Pure  colloids  do  not  carry  an  electric  charge  and 
are  not  conveyed  by  an  electric  current;  their  apparent  charge  depends 
upon  the  nature  of  electrolytes  that  may  be  present.  Traces  of  acid 
and  of  acid  salts  give  it  a  positive  charge,  whereas  alkalis  and  alkaline 
salts  do  the  opposite  (Pauli). 

The  process  of  coagulation  of  proteins,  therefore,  must  depend  upon 

1  Ztsch.  f .  Chem.  Ind.  Koll.,  1907,  2,  3. 


NATURE    AND    PROPERTIES    OF    COLLOIDS  547 

the  neutralization  of  their  electric  charge,  and,  as  previously  stated,  this 
can  be  accomplished  either  by  electrolytes  or  by  colloids: 

(a)  Precipitation  by  electrolytes  is  best  illustrated  by  the  action 
of  a  strong  acid  on  an  albuminous  solution.  The  negatively  charged 
particles  attract  to  themselves  the  positively  charged  hydrogen  ions; 
their  charge  is  now  neutralized,  and  the  force  of  attraction  due  to  their 
surface  tension  is  no  longer  counterbalanced  by  an  electric  repulsion. 
The  particles  are  drawn  together,  form  larger  and  larger  masses,  which 
finally  come  under  the  influence  of  gravity  and  precipitation  takes 
place. 

(6)  Precipitation  of  colloids  by  colloids  is  illustrated  by  the  precipi- 
tation of  albumin  by  acetic  and  ferrocyanic  acids.  The  colloid  must  be 
of  opposite  sign.  As  a  result  of  the  acid  the  particles  acquire  a  positive 
charge,  if  they  are  not  so  charged  already.  This  charge  is  then  neutral- 
ized by  the  colloidal  ferrocyanic  acid  of  negative  sign;  the  surface  ten- 
sion is  no  longer  neutralized  by  an  electric  repulsion,  and  particles  come 
together  to  form  larger  masses  that  are  finally  deposited  as  a  precipitate. 

Instead  of  precipitating  the  other,  an  excess  of  one  colloid  may  act 
in  a  reverse  manner.  For  example,  as  Neisser  and  Friedmann  have 
shown,  a  suspension  of  particles  of  mastic  in  water  (made  by  dropping 
an  alcoholic  solution  in  water)  takes  on  a  negative  charge,  and  can  be 
precipitated  by  positive  colloids  or  ions,  such  as  ferric  chlorid.  If  the 
dose  of  ferric  chlorid  is  increased  gradually,  the  precipitate  becomes 
more  and  more  abundant,  until  an  excess  of  ferric  chlorid  is  present, 
when  the  reaction  ceases  and  the  precipitate  may  be  redissolved.  This 
has  been  explained  on  the  assumption  that  when  two  colloids  of  opposite 
sign  are  mixed,  they  tend  to  fuse  and  form  masses;  the  addition  of  an 
excess  of  either  colloid  tends  to  electrify  the  masses,  causing  mutual 
repulsion  and  possibly  resolution  of  the  masses.  Hence  the  precipitate  is 
soluble  in  an  excess  of  both  substances,  just  as  a  precipitate  is  soluble 
in  an  excess  either  of  precipitin  or  of  its  antigen. 

10.  Absorption  is  the  taking  up  of  dissolved  or  volatile  substances 
by  finely  divided  or  colloidal  bodies.  It  is  a  combination  between  two 
substances  dependent  on  physical  attraction  rather  than  on  chemical 
affinity,  and  taking  place  in  variable  ratios,  rather  than  in  simple  and 
constant  ones,  as  occur  in  a  true  chemical  union.  It  is  believed  by  many 
that  the  two  substances  entering  into  the  phenomenon  of  absorption 
exist  as  such  side  by  side  in  the  compound,  which  is  to  be  regarded 
as  an  intimate  admixture  of  the  two,  rather  than  as  a  new  compound. 

The  lack  of  definite  ratios  by  which  colloids  are  absorbed  has  been 


548         THE    RELATION    OF    COLLOIDS    AND    LIPOIDS   TO    IMMUNITY 

shown  by  Bordet  in  the  amount  of  hemolytic  immune  body  that  can  be 
taken  up  by  a  given  volume  of  corpuscles — i.  e.,  the  amount  varies 
according  to  whether  the  corpuscles  are  added  at  once  or  in  successive 
small  portions.  Thus,  in  one  example,  0.4  c.c.  of  a  hemolytic  serum 
dissolved  0.5  c.c.  of  corpuscles  if  added  at  once;  but  if  0.2  c.c.  of  cor- 
puscles was  added  first  and  successive  amounts  of  0.1  c.c.  then  put  in, 
no  solution  took  place  after  the  one  that  followed  the  addition  of  the  first 
portion.  This  was  explained  by  Bordet  according  to  the  principles  of 
absorption,  this  observer  comparing  it  with  the  absorption  of  a  dye  by 
filter-paper.  While  other  explanations  are  possible,  yet  exactly  analo- 
gous phenomena  may  be  seen  in  the  mutual  absorption  of  colloids  of 
opposite  sign.  Thus,  as  we  have  previously  stated,  the  addition  of 
a  solution  of  an  electropositive  colloid  to  a  solution  of  an  electronegative 
one  tends  to  repel  the  particles,  with  the  formation  of  masses  for  the 
purpose  of  self-protection,  and  in  this  manner  the  process  of  agglutina- 
tion and  precipitation  is  begun.  But  if  a  small  amount  of  a  second  col- 
loid is  added  to  the  same  volume  of  the  others,  new  aggregates  of  the  two 
are  formed  that  are  less  favorable  to  precipitation  and  require  more  of  the 
second  colloid  to  bring  about  complete  precipitation. 

ANALOGY  BETWEEN  THE  REACTIONS  OF  IMMUNITY  AND  COLLOIDAL 

CHEMISTRY 

With  these  few  brief  remarks  on  the  properties  and  nature  of  colloids 
and  the  close  resemblance  of  cellular  protoplasm  and  fluids  to  colloids, 
we  may  consider  briefly  the  apparent  similarity  that  exists  between  the 
colloidal  reactions  and  some  of  the  reactions  of  immunity.  This  is 
especially  pertinent  for  several  reasons :  it  has  been  shown  that  cellular 
protoplasm  is  colloidal  in  nature;  that  antigens  are  certainly  colloidal, 
and  that  antibodies,  while  they  may  or  may  not  be  solutions  of  colloids, 
are,  in  the  final  analysis,  products  of  cellular  activity,  and  therefore 
derived  from  colloidal  solutions. 

1.  Antitoxins. — The  side-chain  theory  of  Ehrlich  was  first  applied 
in  explanation  of  the  principles  of  immunity  as  affording  an  explanation 
of  the  action  of  toxins,  the  formation  of  antitoxin,  and  the  interaction 
between  these.  Ehrlich  has  placed  the  various  phenomena  of  immunity 
upon  a  chemical  basis,  bringing  forward  new  theories  to  explain  the 
various  discrepancies  that  were  found.  For  example,  it  was  soon  found 
that  both  toxin  and  antitoxin  were  unstable,  and  that  neutralization  of 
a  toxin  by  the  addition  of  antitoxin  was  not  a  simple  process,  like  the 
neutralization  of  an  acid  by  an  alkali,  but,  on  the  contrary,  was  likely  to 


ANALOGY    BETWEEN    REACTIONS  549 

be  exceedingly  complicated.  This  was  explained  as  being  due  to  the 
degeneration  of  toxin  into  various  toxoids,  which  were  able  to  neutralize 
antitoxin  without  being  in  themselves  toxic  when  in  a  free  state.  They 
were  likewise  found  to  have  a  greater  affinity  for  antitoxin  than  the  toxin 
itself,  so  that  when  a  toxin  was  tested  and  its  toxicity  determined,  it  was 
discovered  that  more  antitoxin  was  needed  to  neutralize  the  mixture  than 
was  originally  calculated,  because  the  toxoids  took  no  part  in  testing  the 
toxin,  but  were  active  in  uniting  with  antitoxin,  and  in  this  manner 
leaving  true  toxin  unneutralized,  and  therefore  toxic,  unless  an  excess  of 
antitoxin  was  used. 

Analogous  conditions  may  be  observed  among  colloidal  solutions. 
Thus,  Danysz  has  shown  that  more  toxin  is  neutralized  if  antitoxin  is 
added  at  once  than  when  it  is  added  in  successive  doses.  As  stated 
elsewhere,  this  is  explained  by  Ehrlich  upon  the  assumption  that  time 
is  allowed  for  the  degeneration  of  toxin  into  toxoids  to  take  place,  the 
latter  having  a  greater  affinity  for  the  antitoxin.  It  has  been  shown, 
however,  that  in  some  cases  the  addition  of  a  small  amount  of  a  second 
colloid  of  opposite  sign  to  a  colloidal  solution  may  render  the  solution 
more  stable  and  protect  it  from  precipitation  by  an  excess  of  the  second 
substance.  Similarly,  the  amount  of  colloid  necessary  to  precipitate 
a  constant  amount  of  another  colloid  is  reduced  to  a  minimum  if  the 
addition  is  made  at  once,  and  is  rendered  much  greater  if  the  colloid 
added  is  made  slowly  in  small  amounts,  an  interval  being  allowed  to 
elapse  after  each  addition.  This  is  closely  analogous  to  the  Danysz 
reaction,  and  explains  the  latter  as  being  due,  when  antitoxin  is  added 
slowly  to  toxin,  to  the  formation  of  transitional  compounds  of  toxin 
and  antitoxin  of  diverse  nature,  requiring  more  antitoxin  for  complete 
neutralization  of  the  toxin  than  if  the  antitoxin  were  added  at  once  and 
in  one  dose. 

The  difference  between  the  L0  and  L+  dose  of  a  toxin  also  has  an  an- 
alogy in  the  reaction  of  simple  colloidal  substances.  Thus,  Bilty  has  used 
ferric  hydroxid,  which  neutralizes  arsenic  trioxid  (the  antidote  for  acute 
arsenical  poisoning),  and  found  that  the  addition  of  one  lethal  dose  of 
arsenic  to  a  neutral  mixture  of  the  two  did  not  render  the  mixture  toxic, 
but  that  several  lethal  doses  were  required,  just  as  it  is  necessary  to  add 
several  instead  of  one  lethal  dose  of  diphtheria  toxin  to  the  LO  dose. 

Thus  it  would  appear  that  the  neutralization  of  a  toxin  by  an  anto- 
toxin  has  analogies  among  the  known  and  simple  colloidal  reactions. 
One  objection  to  placing  the  toxin-antitoxin  reaction  upon  a  colloidal 
basis  is  that  both  have  the  same  electric  charge,  i.  e.,  both  move  toward 


550         THE    RELATION    OF    COLLOIDS    AND    LIPOIDS    TO    IMMUNITY 

the  cathode,  and,  as  we  have  seen,  for  the  neutralization  and  precipita- 
tion of  colloids  the  solution  of  colloids  should  be  of  opposite  sign.  It 
must  be  remembered,  however,  that  toxins  and  antitoxins  react  in  very 
complex  fluids  containing  other  substances  consisting  of  both  colloids 
and  electrolytes,  and  until  the  electric  charge  of  these  in  pure  form  is 
determined,  the  apparent  similar  electric  charge  of  toxin  and  antitoxin 
can  hardly  outweigh  the  otherwise  remarkable  analogy  it  bears  to  col- 
loidal reactions. 

2.  Agglutinins  and  Precipitins. — Various  theories  in  explanation  of 
the  phenomenon  of  agglutination  have  been  described  in  a  previous 
chapter.  The  theory  of  Bordet  appears  to  be  best,  and  is  based  upon 
certain  principles  of  colloidal  chemistry.  When  bacteria  are  suspended 
in  a  fluid  free  from  salt,  agglutination  does  not  take  place  because  the 
bacteria  carry  a  similar  negative  charge  of  electricity.  When,  however, 
ions  of  positive  charge  are  added,  as,  e.  g.,  sodium  chlorid,  the  bacteria  or 
other  cells  are  repelled  and  coalesce  to  form  masses,  according  to  the 
laws  of  surface  tension,  in  an  effort  to  protect  themselves.  Larger 
masses  may  be  formed  that  finally  come  within  the  influence  of  gravity 
and  are  deposited  at  the  bottom  of  the  test-tube.  According  to  the 
same  laws,  the  addition  of  agglutinin  removes  the  negative  charge  of 
bacteria  or  other  cells,  with  the  consequent  formation  of  clumps  and 
masses.  Similar  phenomena  may  be  observed  in  the  precipitation  of 
colloidal  suspensions  of  clay  in  distilled  water  by  the  addition  of  a  salt. 

Solutions  of  inorganic  colloids,  as,  for  example,  that  of  silicic  acid, 
may  agglutinate  red  corpuscles;  bacteria,  such  as  suspensions  of  typhoid 
and  colon  bacilli,  may  be  agglutinated  by  solutions  of  the  ferric  salts. 

Just  as  an  excess  of  one  colloid  solution  will  charge  masses  of  the 
other,  resulting  in  a  repelling  action  and  breaking  up  of  the  agglutinated 
clumps,  so  the  addition  of  an  excess  of  agglutinin  is  found  to  prevent 
agglutination  or  to  give  but  a  slight  reaction.  This  phenomenon  has 
been  explained,  according  to  Ehrlich's  side-chain  theory,  as  due  to  the 
presence  of  agglutinoids  that  have  a  great  affinity  for  the  bacteria  and 
unite  with  them  without  being  active  in  the  free  state,  owing  to  a  loss 
of  the  agglutinophore  portion  of  the  molecule.  Each  cell  united  with 
an  agglutinoid  is  one  cell  less  to  undergo  agglutination  by  agglutinin, 
and  accordingly  in  weak  dilutions  of  serum  agglutination  is  feeble  or 
absent  whereas  hi  higher  dilutions  the  phenomenon  may  be  clearly 
observed. 

Other  explanations  of  the  action  of  agglutinins  and  precipitins, 
based  upon  colloidal  reactions,  have  been  advanced.  Thus  Neisser  and 


ANALOGY    BETWEEN    REACTIONS  551 

Friedmann  have  shown  that  suspensions  of  mastic  may  be  ''protected" 
against  the  precipitating  action  of  ferric  hydroxid  by  the  addition  of  a 
small  amount  of  organic  colloid,  such  as  serum,  leech  extract,  or  extract 
of  typhoid  bacilli,  regardless  of  whether  this  colloid  is  charged  posi- 
tively or  negatively  or  is  neutral.  The  aforenamed  observers  believe 
that  normal  bacteria  may  be  surrounded  by  a  similar  protective  envelop 
that  prevents  the  agglutinating  action  of  substances  of  opposite  sign. 
The  action  of  agglutinin,  therefore,  would  be  to  remove  this  layer,  so 
that  the  ions  of  opposite  electric  charge  can  unite  with  the  bacteria  and 
bring  about  their  agglutination.  This  may  be  an  explanation  of  the 
role  of  salts  in  the  phenomenon  of  agglutination,  the  agglutinins  remov- 
ing the  protecting  envelops  and  the  salt  furnishing  the  ions  of  opposite 
charge  that  bring  about  agglutination. 

Owing  to  the  fact  that  a  discrepancy  arises  here  for  the  reason  that 
emulsions  of  red  corpuscles  are  agglutinated  by  both  positive  and 
negative  colloids  (ferric  hydroxid  and  cuprum  ferrocyanid),  Girard, 
Mangin  and  Henri  have  given  the  following  explanation  of  agglutina- 
tion: When  a  red  corpuscle  is  suspended  in  a  fluid,  various  salts,  es- 
pecially the  sulphates  of  magnesium  and  calcium,  are  diffused,  which 
tends  to  facilitate  the  precipitation  of  negative  and  positive  colloids, 
so  that  each  corpuscle  comes  to  be  surrounded  by  a  layer  of  precipitated 
colloid  material.  This  zone  of  precipitated  colloids  of  either  negative 
or  positive  charge  determines  agglutination  in  the  presence  of  a  colloid 
solution  of  opposite  charge,  such  as  agglutinin  or  inorganic  colloids 
(silicic  acid,  etc.). 

3.  Hemolysins. — Reference  has  been  made  elsewhere  to  the  original 
observations  of  Bordet,  showing  that  red  corpuscles  may  absorb  much 
more  hemolytic  antibody  than  is  necessary  to  bring  about  their  lysis, 
and  that  this  absorption  is  analogous  to  colloidal  absorption. 

Inorganic  colloidal  solutions,  such  as  that  of  silicic  acid,  may  produce 
hemolysis  of  red  blood-corpuscles,  e.  g.,  those  of  the  rabbit.  Its  action 
is  manifested  in  extremely  small  doses.  It  is  rendered  inert  by  heat, 
and  gradually  deteriorates  at  room  temperature.  Furthermore,  this 
inorganic  colloid  possesses  some  of  the  properties  of  a  serum  hemolysin; 
thus  mice  red  corpuscles  that  have  been  agglutinated  by  colloidal  silicic 
acid  are  dissolved  by  traces  of  lecithin  or  of  fresh  serum,  but  not  by  serum 
that  has  been  heated  to  60°  C.  (inactivated).  An  excess  of  silicic  acid 
tends  to  prevent  hemolysis,  which  is  another  example  of  the  action  of  an 
excess  of  one  colloidal  solution  upon  another  of  opposite  sign. 

Probably  saponin  hemolysis  and  the  influence  of  fatty  substances, 


552         THE    RELATION    OF    COLLOIDS    AND    LIPOIDS    TO    IMMUNITY 

such  as  lecithin  and  the  fatty  acids,  upon  the  phenomenon  of  hemolysis, 
are  closely  related  to,  or  to  be  explained  by,  the  action  of  organic  colloidal 
solutions. 


THE  COMPLEMENT-FIXATION  TEST  AS  A  COLLOIDAL  REACTION 
Many  observations  support  the  view  that  complement  fixation  by  a 
specific  antigen  and  its  antibody  is  really  complement  absorption  by  a 
precipitate  that  forms  when  antigen  and  antibody  are  mixed.  As 
previously  stated,  all  antigens  are  protein  in  character.  While  there  is 
some  evidence  to  show  that  lipoids,  and  even  carbohydrates,  may  act  as 
antigens,  there  is  no  doubt  but  that  the  chief  antigenic  principles  of  any 
antigen  are  of  protein  structure;  hence  when  mixed  with  an  immune 
serum  containing  specific  antibodies,  it  is  believed  that  an  invisible 
precipitate  is  formed  that  absorbs  the  complement.  With  serum  anti- 
gens the  quantity  of  protein  is  so  large  that  a  precipitate  can  readily  be 
seen  (the  precipitin  test).  A  serum  antigen  and  its  antibody  may, 
however,  be  so  highly  diluted  that,  when  mixed,  a  precipitate  is  not 
visible,  although  complement  may  be  fixed  (complement-fixation  test 
for  the  differentiation  of  proteins).  Moreschi  and  Gay  have  contended 
for  many  years  that  complements  may  become  entangled  and  absorbed 
in  such  precipitates.  Reasoning  on  the  basis  of  the  colloidal  theory, 
it  is  possible  that  transition  compounds  of  very  diverse  nature  are 
formed  when  antigen,  antibody,  and  a  complement  are  mixed.  This 
view  on  the  action  of  complements  and  anti-complements  is  supported 
by  numerous  investigators  who  have  examined  the  question  from  the 
standpoint  of  colloidal  reactions.  Thus  in  a  complement-fixation  test  a 
mixture  of  antigen,  antibody,  and  a  complement  in  definite  proportions 
results  in  the  formation  of  new  compounds  of  opposite  electric  charge, 
which  tend  to  aggregate  in  masses  (although  these  may  be  so  small  as 
to  be  invisible)  and  reduce  their  surface  tension  in  just  the  same  manner 
as  agglutination  and  precipitation  are  brought  about  after  the  colloidal 
theories.  When  corpuscles  and  hemolytic  antibody  are  subsequently 
added,  hemolysis  does  not  occur  because  free  complement  is  absent. 

A  process  similar  to  complement  absorption  by  a  specific  antigen 
and  its  antibody  is  the  Wassermann  reaction.  According  to  the  col- 
loidal theories,  this  reaction  may  be  explained  as  due  to  the  formation 
of  an  invisible  precipitate  by  interaction  between  some  substance  in 
the  serum  of  a  luetic  person  (probably  in  the  nature  of  an  altered  globu- 
lin), complement,  and  lipoidal  substances  contained  in  an  alcoholic  or 


THE    RELATION    OF    LIPOIDS    TO    IMMUNITY  553 

watery  extract  of  a  normal  or  a  diseased  organ.  This  view  is  supported 
by  the  fact  that  euglobulin  is  known  to  be  generally  increased  in  the 
body  fluids  of  syphilitics,  and  by  analogy  with  the  various  precipitin  tests 
that  have  been  devised  for  the  diagnosis  of  syphilis,  as,  for  example,  the 
reaction  of  Forges  and  Meier,  which  is  dependent  upon  the  appearance 
of  a  precipitate  when  luetic  serum  is  mixed  with  an  emulsion  of  lecithin 
or  sodium  glycocholate,  etc.  The  exact  nature  of  the  antibody  in 
syphilitic  serums  that  forms  these  new  compounds  with  lipoids  and 
complement,  resulting  probably  in  the  absorption  of  complement,  is 
unknown.  It  is  most  likely  in  the  nature  of  a  globulin,  its  main  char- 
acteristic being  the  power  it  possesses  of  reacting  with  lipoids.  Schmidt l 
ascribes  the  reaction  to  the  physicochemical  properties  of  the  globulins 
of  the  syphilitic  serum,  which  he  believes  possess  a  greater  affinity  for 
the  colloids  of  the  antigen  than  do  normal  globulins.  This  view  is 
supported  by  the  common  observation  that  the  turbidity  of  the  antigen 
emulsion  is  closely  related  to  its  efficiency,  since  clear  solutions  are  less 
active.  Since  various  lipoidal  substances  may  be  employed,  the  Was- 
sermann  reaction  can  not  be  regarded  as  specific  in  the  immunologic 
sense,  although  practically  it  is  highly  specific,  as  similar  conditions  are 
to  be  found  in  only  two  other  diseases  with  any  degree  of  regularity, 
namely,  frambesia  and  tuberous  leprosy. 


THE  RELATION  OF  LIPOIDS  TO  IMMUNITY 

It  is  becoming  more  and  more  evident  that  lipoids  bear  an  important 
relation  to  various  immunologic  processes,  especially  to  certain  cytolytic 
phenomena. 

As  stated  in  Chapter  VIII,  the  results  of  some  researches  that  go  to 
show  certain  lipoids  and  lipoidal  substances  may  act  as  true  antigens 
and  produce  antibodies.2  This,  however,  has  not  been  definitely  proved, 
and  while  it  is  of  great  importance,  is  not  necessarily  pertinent  to  the 
subject  in  hand.  As  the  relation  of  lipoids  to  various  immunologic 
processes  has  frequently  been  described  in  earlier  chapters,  as,  e.  g., 
where  the  role  of  lipoids  in  venom  hemolysis,  in  the  Wassermann  syphilis 
reaction,  and  in  the  various  preoipitin  reactions  in  syphilis  were  con- 
sidered, a  brief  resume  may  be  of  service  in  directing  attention  to  this 
important  and  particular  phase  of  immunity. 

1  Zeit.  f.  Hygiene,  1811,  69,  513. 

2  Bibliography  on  Lipoids  and   Immunity  given  by  Landsteiner,  Kolle  and 
Wassermann's  Handbuch,  1913,  2,  1240.     Also  review  of  literature  by  Landsteider, 
Jabresb.  Immunitat,  1910,  6,  209. 


554        THE   RELATION    OF    COLLOIDS   AND    LIPOIDS   TO    IMMUNITY 

Relation  of  Lipoids  to  Hemolysis. — (a)  From  the  standpoint  of 
immunity,  venom  hemolysis  is  of  peculiar  interest  as  indicating  the  pos- 
sible important  relation  of  lipoids  to  hemolytic  complement.  Granting 
that  venom  contains  a  hemolytic  amboceptor  (Flexner  and  Noguchi), 
the  complementing  substance  must  be  derived  from  the  corpuscles,  and, 
according  to  Kyes,  this  complementary  agent  is  represented  in  lecithin. 
Kyes  was  able  to  produce  what  he  considers  are  compounds  of  the  hemo- 
lysin  with  lecithin,  namely,  "lecithids."  Whether  these  "lecithids" 
are  true  compounds  of  hemolysins  and  corpuscular  lecithin  or  simply  the 
active  hemolytic  products  of  the  cleavage  of  lecithin  by  ferments  con- 
tained in  the  venom,  is  at  present  unknown.  Noguchi  and  Lieberman 
have  shown  that  not  only  lecithin,  but  soap  as  well,  especially  unsatu- 
rated  fatty  acids,  and  probably  protein  compounds  of  soaps  and  lecithin, 
may  act  as  the  hemolytic  complement  and  activate  the  hemolysin  of  the 
venom.  Lipoids  from  bacteria  and  trypanosomes  have  been  found  to 
possess  similar  properties.  Hemolytic  lipoids  have  been  secured  from 
serum,  and  the  complementary  activity  of  a  fresh  normal  serum  may  be 
destroyed  by  fat  solvents,  e.  g.,  ether.  While  other  investigators  have 
not  been  able  to  confirm  Noguchi's  attempts  to  produce  an  artificial 
complement  of  fatty  substances  of  exactly  the  same  properties  as  serum 
complement,  this  work  indicates  most  strongly  the  close  relation  of 
serum  complement  to  lipoids. 

(b)  The  hemotoxic  activity  of  various  toxins  is  probably  dependent 
largely  upon  their  action  on  the  lipoids  of  red  corpuscles.  The  saponin 
substances, l  a  group  closely  related  to  glucosids,  and  found  in  at  least 
46  different  families  of  plants,  are  strongly  hemolytic.  Ransom2  has 
found  that  an  ethereal  extract  of  red  corpuscles  contains  a  substance 
that  inhibits  saponin  hemolysis.  This  substance  consists  largely  of 
cholesterin,  and  it  is  the  presence  of  cholesterin  in  normal  serum  that 
inhibits  saponin  hemolysis.  This  may  be  demonstrated  experimentally 
by  adding  cholesterin  to  a  solution  of  a  saponin.  Noguchi 3  has  shown 
that  lecithin  does  not  possess  the  same  antihemolytic  action  on  saponin. 
It  would  appear,  therefore,  that  saponin  causes  hemolysis  by  combining 
with,  altering,  or  dissolving  the  lipoids  of  the  stroma  of  corpuscles.  The 
resistance  of  corpuscles  to  saponin  hemolysis  varies  in  certain  diseases, 
being  especially  low  in  jaundice  (McNeil)4. 

1  Complete  literature  on  saponin,  see  Robert  "Die  Saponinsubstanzen,"  Stutt- 
gart, 1904. 

2  Deut.  med.  Wochenschr.,  1901,  27,  194. 

3  Univ.  of  Penna.  Med.  Bull.,  1902,  15,  327. 

4  Jour.  Path,  and  Bact.,  1910,  15,  56. 


THE   RELATION   OF   LIPOIDS   TO   IMMUNITY  555 

While  saponins,  solanins,  phallin,  and  other  vegetable  poisons  are  of 
relatively  simple  chemical  composition  and  quite  unlike  proteins, 
enzymes,  or  toxins,  it  is  possible  that  bacterial  and  vegetable  hemo- 
toxins,  such  as  tetanolysin,  abrin,  ricin,  crotin,  and  robin,  may  produce 
their  effects  by  a  similar  action  on  the  lipoids  of  the  erythrocytes. 
Noguchi  has  shown  that  cholesterin  inhibits  the  action  of  tetanolysin. 
Landsteiner  and  Bottori  have  found  that  protagon,  a  brain  lipoid, 
possesses  the  property  of  binding  tetanus  toxin,  which  indicates  that 
this  toxin  may  produce  its  effects  by  some  action  upon  the  lipoids  of 
nerve-cells. 

(c)  The  important  relation  of  lipoids  to  the  Wassermann  reaction  and 
certain  precipitin  or  floccule-forming  reactions  (Klausner,  Porges-Meier, 
Hermann-Perutz)  has  been  mentioned  repeatedly.  Just  what  role  the 
lipoids  play  in  these  phenomena  is  not  known.  While  the  globulins  of 
syphilitic  serums  are  strongly  suspected  of  being  concerned  in  these 
processes,  their  relation  is  not  clear.  Klausner1  now  believes  that  the 
precipitate  that  forms  when  distilled  water  is  added  to  syphilitic  serum 
is  due  to  the  high  lipoid  content. 

LANGE'S  COLLOIDAL  GOLD  REACTION 

Principles. — According  to  the  exhaustive  studies  of  Zsigmondy2  on 
metallic  colloids  a  solution  of  a  protein  will  precipitate  colloidal  gold 
in  the  absence  of  an  electrolyte.  An  electrolyte,  as  sodium  chlorid, 
will  in  certain  concentrations  precipitate  the  colloidal  gold  itself;  if 
proteins  are  present  this  precipitation  is  inhibited.  The  degree  of 
protection  afforded  has  been  found  specific  for  each  protein,  and  is  ex- 
pressed in  terms  of  milligrams  of  the  protein  capable  of  protecting  5  c.c. 
of  colloidal  gold  against  0.5  c.c.  of  a  10  per  cent,  solution  of  sodium 
chlorid. 

Lange3  endeavored  to  distinguish  between  normal  and  luetic  sera 
by  this  means  on  the  basis  of  disturbances  in  syphilis,  but  failed;  he 
then  sought  to  measure  the  protein  content  of  cerebrospinal  fluid  by 
the  degree  of  precipitation  of  gold,  but  failed  again,  because  he  used 
distilled  water  as  a  diluent,  thereby  throwing  the  protein,  particularly 
the  globulins,  out  of  solution,  and  rendering  them  inert.  When,  how- 
ever, he  used  a  0.4  per  cent,  solution  of  sodium  chlorid  as  a  diluent  it 
was  found  that  the  proteins  were  not  precipitated  and  that  this  amount 
of  salt  was  too  weak  to  precipitate  the  colloidal  gold. 

1  Biochem.  Zeit.,  1912,  47,  36. 

2  Ztsch.  f.  Anal.  Chem.,  1901,  xl,  697. 

3  Berl.  klin.  Wchnschr.,  1912,  xlix,  897;  Ztschr.  f.  Chemotherap.,  1913,  1,  44. 


556         THE    RELATION    OF    COLLOIDS    AND    LIPOIDS   TO    IMMUNITY 

It  was  now  possible,  according  to  Lange,  to  measure  the  protein 
content  of  spinal  fluid  according  to  the  degree  of  precipitation,  and  very 
interesting  and  practical  results  have  been  secured,  although  it  cannot 
be  stated  as  proved  that  the  precipitation  is  due  entirely  to  protein, 
particularly  since  it  has  been  shown  that  the  globulins  may  actually 
protect  colloidal  gold  against  precipitation.  Zaloziecki1  regards  the 
reaction  as  a  form  of  immunity  reaction;  Jaeger  and  Goldstein2  consider 
it  purely  physical  and  probably  of  an  electric  nature. 

Preparation  of  Colloidal  Gold. — The  preparation  of  colloidal  gold 
is  frequently  an  exceedingly  troublesome  procedure,  and  the  success 
of  the  test  depends  upon  a  satisfactory  preparation.  The  method  which 
I  shall  briefly  describe  here  is  after  that  of  Miller,  Brush,  Hammers, 
and  Felton,3  which  I  have  found  to  yield  fairly  constant  and  satisfac- 
tory products. 

The  glass-ware  (beakers,  pipets,  and  test-tubes)  must  be  absolutely  dean. 
They  may  be  washed  in  hot  water  with  ivory  soap;  rinsed  in  tap-water 
for  five  minutes;  placed  in  hot  bichromate  cleaner  for  half  an  hour; 
rinsed  in  tap-,  and  finally  triple  distilled  water.  The  beakers  should  be 
used  at  once;  the  pipets  and  test-tubes  are  to  be  dried  in  a  hot-air  oven. 
Thermometers  should  be  cleansed  in  a  similar  manner. 

It  is  necessary  to  use  water  triply  distilled  in  an  apparatus  free  of 
rubber  connections.  The  reagents  should  be  freshly  prepared  of  the  best 
products  obtainable. 

1.  Heat  1000  c.c.  of  triply  distilled  water  over  a  good  Bunsen  burner 
in  a  prepared  beaker  with  a  thermometer. 

2.  At  60°  C.  add  10  c.c.  of  a  1  per  cent,  solution  of  Merck's  gold 
chlorid  c^stals  in  triply  distilled  water  and  7  c.c.  of  a  2  per  cent,  solu- 
tion of  Merck's  blue  label  potassium  carbonate  in  triply  distilled  water. 

3.  At  80°  C.,  while  stirring  briskly,  add  10  drops  of  a  1  per  cent,  solu- 
tion of  Merck's  blue  label  oxalic  acid  crystals  in  triply  distilled  water. 

4.  At  90°  C.  remove  the  burner  and,  while  stirring,  add  5  c.c.  of  a 
solution  of  1  c.c.  of  Merck's  highest  purity  formaldehyd  in  40  c.c.  of 
triply  distilled  water,  or  enough  to  produce  an  initial  pink  color. 

5.  The  solution  must  be  neutral  in  reaction  when  used,  and  for  this 
purpose  is  tested  with  a  1  per  cent,  solution  of  alizarin  red  in  50  per  cent, 
alcohol.    With  this  indicator  the  neutral  point  is  a  brownish-red  tint; 
an  acid  solution  gives  a  lemon-yellow,  and  an  alkaline  solution  a  pur- 
plish-red, color. 

1  Deutsch.  ztsch.  f.  Nervenhl.,  1913,  xlvii,  783. 

2  Ztsch.  f.  d.  ges.  Neur.  u.  Psych.,  1913,  xvi,  219. 

3  Bull.  Johns  Hopkins  Hosp.,  1915,  xxvi,  391. 


THE    RELATION    OF   LIPOIDS   TO    IMMUNITY  557 

To  10  c.c.  of  the  colloidal  gold  in  a  clean  beaker  add  2  drops  of 
indicator.  If  it  is  acid,  titrate  to  the  neutral  point  with  -f$  NaOH;  if 
alkaline,  with  ^  Hcl.  Calculate  the  amount  to  be  added  for  the  amount 
of  colloidal  gold  solution  at  hand  and  neutralize  with  normal  or  deci- 
normal  solutions  of  acid  or  alkali  as  required. 

The  following  are  suitable  standards  for  a  good  colloidal  gold 
product: 

(a)  Absolutely  transparent  and  of  a  brilliant  red  orange  or  salmon- 
red  color. 

(6)  Five  c.c.  of  the  solution  must  be  completely  precipitated  in  one 
hour  by  1.7  c.c.  of  a  1  per  cent,  solution  of  sodium  chlorid  in  distilled 
water. 

(c)  The  solution  must  be  neutral  in  reaction  when  used. 

(d)  The  solution  must  not  produce  a  reaction  greater  than  a  No.  1 
with  normal  cerebrospinal  fluid,  and  must  give  a  typical  reaction  curve 
with  a  known  pare  tic  fluid. 

The  Test. — 1.  Arrange  eleven  clean,  dry  test-tubes  in  a  row;  put 
1.8  c.c.  of  fresh,  sterile  0.4  per  cent.  NaCl  solution  into  the  first  tube 
and  1  c.c.  in  the  following  ten. 

2.  With  a  clean,  dry  pipet  add  0.2  c.c.  of  a  blood-free  cerebrospinal 
fluid  to  the  first  tube  and  mix;  transfer  1  c.c.  from  the  first  to  the  second 
tube,  mix,  and  proceed  in  this  manner  up  to  and  including  the  tenth  tube, 
from  which  1  c.c.  is  discarded.    The  eleventh  tube  is  the  control  and 
contains  no  cerebrospinal  fluid.     The  dilutions  now  range  from  1  to  10 
to  1  to  5120. 

3.  Add  to  each  tube  5  c.c.  of  colloidal  gold;  mix  and  stand  aside  at 
room  temperature  over  night;  the  readings  are  made  the   next  day 
and  recorded  by  numbers  according  to  the  following  scheme: 

5   =  complete  precipitation  (water  clear). 

4   =   pale  blue. 

3   =  blue. 

2   =   lilac  or  purple. 

1   =  red-blue. 

0   =  no  change. 

Types  of  Reactions. — 1.  Normal  fluid  produces  no  changes  at  all 
or,  at  most,  a  No.  1  change  in  the  first  tube  of  the  series. 

2.  The  typical  reaction  is  observed  in  general  paresis,  giving  com- 
plete precipitation  in  the  first  four  to  eight  tubes  of  the  series,  with 
changes  of  color  in  most  of  the  remaining  ones,  as,  for  example. 
5555542100,  and  constituting  the  "paretic  curve"  of  Miller  and 


558         THE    RELATION    OF   COLLOIDS   AND    LIPOIDS   TO    IMMUNITY 


Levy.1    A  similar  curve  is  usually  found  in  taboparesis.     See  table  20a 
and  Fig.  122. 

TABLE  20a-SHOWING  THE  FOUR  COMMON  TYPES  OF  COLLOIDAL 

REACTIONS 


Color  Reactions 

Numbers  used 
for  designating 
Color  Reactions 

Dilutions  of  J 

oinal  Fluid  with  QA-  1 

oer  cent.Nacl. 

J.-10 

1:20 

1:40 

7:80 

1:160 

7:320 

1:640 

1:1280 

J.Z560 

1:512.0 

Complete  clecoloradtion 

S 

— 

/\ 

\ 

Pale  blue 

± 

\ 

/ 

S 

\ 
\. 

Blue 

3 

.... 

\ 

'•/ 

/ 

\ 

Lilac  or  purple 

Z 

•*°^.-' 

.^'- 

\ 

\      \ 

\ 
\ 

fled—  blue 

7 

..-•••'' 

;>' 

\ 

\ 

\ 

\ 

Brilliant  Red  orange 

0 

-:: 

-"—- 

—  x^ 

\ 

-—  - 

--.- 

No  reaction;  normal  fluid. 
Paretic  Reaction  Type. 
(Fig.  122.) 


Luetic  Reaction  Type  (Fig.  122). 
Meningitis  Reaction  Type. 
(Verschiebung  nach  oben.) 


3.  The  cerebrospinal  fluid  in  tabes  dorsalis  usually  produces  the 
"luetic  zone"  curve  of  a  No.  4  intensity,  as,  for  example,  4445542000; 
precipitation  being  partial  in  the  first  two  or  three  tubes,  then  becoming 
complete,  and  gradually  returning  to  normal  through  the  balance  of  the 
series.    The  changes,  however,  are  not  constant  or  characteristic. 

4.  The  fluids  in  cerebrospinal  syphilis  usually  yield  weak  reactions 
of  the  "luetic  zone"  type.     In  tertiary  syphilis  without  symptoms 
referable  to  the  central  nervous  system  similar  reactions  are  frequently 
observed. 

5.  Fluids  from  cases  of  purulent  or  tuberculous  meningitis  may 
give  reactions  which  are  usually  maximal  in  the  higher  dilutions,  the 
"meningitic  zone"  type  and  so-called  "Verschiebung  nach  oben."     In 
acute  anterior  poliomyelitis  the  cerebrospinal  fluid  frequently  yields  reac- 
tions similar  to  the  "luetic"  and  "meningitic"  zone  types  of  reactions. 

Practical  Value  of  the  Colloidal  Gold  Test.— The  reports  of  a  very 
large  number  of  investigators,  including  Lange,2  de  Cruris  and  Frank,3 
de  Cruris  and  Eberhardt,4  Grulee  and  Moody,5  Eicke,  Jaeger6  and 
Goldstein,7  Klienberger,8  Zaloziecki,9  Kaplan,10  Miller  and  Levy,11 

1  Bull.  Hopkins  Hosp.,  1914,  xxv,  123. 

2  Ztsch.  f.  Chem.  u.  v.  Gehete.,  1913,  1,  44;  Berl.  klin.  Wchnschr.,  1912,  xlix,  897. 

3  Munch.  Med.  Wchnschr.,  1915,  Ixi,  1209. 

4  Munch.  Med.  Wchnschr.,  1914,  61,  1216. 

5  Amer.  Jour.  Dis.  Child.,  1915,  ix,  19. 

6  Munch.  Med.  Wchnschr.,  1913,  Ix,  2713.  7  Loc  cit. 
8  Arch.  f.  Psych.,  1911,  xlviii,  264.                                                     9  Loc  cit. 

10  Ztsch.  f.  d.  ges.  Neurol.  u.  Psych.,  1915,  xxvii;  Jour.  Amer.  Med.  Assoc.,  1914, 
Ixii,  511,  246. 

11  Loc  cit. 


THE    RELATION    OF    LIPOIDS    TO    IMMUNITY  559 

Swalm  and  Mann,1  Lee  and  Hiriton,2  Weston,  Darling  and  Newcomb,3 
Solomon  and  Welles,4  Miller,  Brush,  Hammers  and  Felton5,  and  others, 
show  that  under  proper  conditions  the  colloidal  gold  test  is  highly 
specific  and  of  value  in  the  diagnosis  of  general  paresis. 

A  paretic  colloidal  gold  reaction  with  the  cerebrospinal  fluid  of  a 
luetic  person  may  be  the  first  sign  of  an  incipient  paresis. 

A  paretic  reaction  with  the  cerebrospinal  fluid  of  a  luetic  individual 
should  be  regarded  as  of  grave  import;  while  positive  symptoms  of 
paresis  may  not  be  present,  intensive  antiluetic  treatment  may  serve 
to  arrest  the  disease. 

The  colloidal  gold  reactions  in  tabes  dorsalis  and  cerebrospinal 
syphilis  are  not  characteristic  of  these  diseases,  and  at  most  may  yield 
the  luetic  zone  type  of  reaction,  and  thereby  prove  of  value  in  confirming 
a  doubtful  diagnosis. 

THE  EPIPHANIN  REACTION 

Principle. — This  reaction  is  based  upon  the  observation  made  by 
Weichardt6  in  1908;  he  found  that  diffusion  is  accelerated  when  dif- 
ferently colored  solutions  of  antigen  and  its  specific  antibody  are  brought 
together.  Changes  in  diffusion  are  associated  with  changes  in  the 
surface  tension,  both  of  which  depend  on  a  change  in  the  osmotic  pres- 
sure. This  is  the  principle  made  use  of  by  Ascoli  in  his  miostagmin 
reaction,  which  will  be  described  further  on. 

Later  Weichardt  made  the  reaction  more  accessible  to  practical  use 
by  introducing  into  the  solution  of  serums  and  antigen  a  system  composed 
of  sulphuric  acid  and  barium  hydroxid,  together  with  certain  catalytic 
agents.  Using  phenolphthalein  as  an  indicator,  he  could  show  that 
fresh  serums  in  high  dilutions  alter  the  surface  tension  of  the  finely 
divided  barium  sulphate  particles  by  their  colloidal  action,  so  as  to  in- 
crease the  absorption  of  H-ions,  thus  rendering  the  solution  more  alkaline. 

This  phenomenon  has  been  utilized  by  Weichardt,  under  the  name 
of  "epiphanin  reaction,"  to  determine  the  occurrence  of  such  interac- 
tion of  antigen  and  antibody.  The  reaction  probably  depends  upon 
physicochemical  principles  of  absorption,  but  the  exact  nature  of  the 

1  N.  Y.  Med.  Jour.,  1915,  xi,  719. 

2  Amer.  Jour.  Med.  Sci.,  1914,  cxlviii,  33. 

3  Amer.  Jour.  Insan.,  1915,  Ixxi,  773. 

4  Boston  Med.  and  Surg.  Jour.,  1914,  clxxi,  886;  ibid.,  1915,  clxxii,  398. 
6  Loc  cit. 

•°  Berl  klin  Wochenschr,  1908,  No.  20;  Centralbl.  f.  Bakteriol.,  xliii,  143-  ibid 
19U   No  4G1154r'      Immumt*tsforsch->  1910>  ™>  651;  Deutsch.  med.  Wochenschr.'; 


560         THE    RELATION    OF    COLLOIDS   AND    LIPOIDS   TO    IMMUNITY 

change  is  not  yet  understood.    The  reaction  is  based  upon  the  following 
generalizations : 

1.  Solutions  containing  colloids — i.  e.,  antigen  alone,  antiserum  alone, 
or  antigen  plus  non-specific  antiserum  in  certain  dilutions — act  in  the 
foregoing  system  by  shifting  the  phenolphthalein  end-point  (the  point  of 
neutralization  when  acid  and  alkali  are  brought  together  in  the  presence 
of  this  indicator)  in  the  sense  of  increased  OH-ions  (pink  color) . 

2.  Specific  antigens  can  inhibit  the  activity  of  their  specific  antise- 
rums,  the  specific ,  antigen-antibody  combination  then  becoming  evident 
in  vitro  by  a  shift  of  the  end-point  in  the  sense  of  increased  H-ion  con- 
centration (light  color). 

Specificity. — The  specificity  of  the  reaction  has  been  confirmed  by  a 
number  of  investigators  who  used  the  test  for  the  identification  of  a 
host  of  antigen-antibody  combinations  in  vitro.  The  underlying  prin- 
ciples have  been  confirmed  by  Kraus  and  Amiradzibi,1  Schroen,2 
Seifert,3  Mosbacher,4  and  others.  The  reaction  has  been  applied  to  a 
study  of  various  antigens  and  their  antibodies,  such  as  diphtheria  toxin, 
tetanus  toxin,  typhoid  and  tubercle  bacilli,  tumor  extracts  and  placenta 
extracts  by  Weichardt;  extracts  of  sjrphilitic  livers  and  serums  of 
syphilitic  patients  by  Seifert,  Keidel  and  Hurwitz.5 

Technic. — The  technic  of  this  reaction  has  been  modified  from  time 
to  time.  The  method  here  given  is  essentially  the  latest  given  by 
Weichardt,6  slightly  modified  by  Keidel  and  Hurwitz. 

Five  constituents  enter  into  the  test: 

1.  The  Antigen. — This  is  an  alcoholic  extract  of  syphilitic  liver, 
prepared  in  exactly  the  same  manner  as  for  performing  the  Wassermann 
reaction.     High  dilutions  of  the  antigen,  ranging  from  1 : 100  to  1 : 10,000, 
are  prepared  with  normal  salt  solution.     As  in  the  Wassermann  re- 
action, not  every  antigen  is  satisfactory,  a  point  that  can  be  determined 
only  by  making  preliminary  tests. 

2.  The  patient's  serum  should  be  fresh,  unheated,  and  highly  diluted, 
the  dilutions  ranging  from  1 : 100  to  1 : 10,000,000.     Usually  it  is  better  to 
use  higher  than  lower  dilutions.     When  too  concentrated  solutions  of 
serums  and  of  antigen  are  used,  erroneous  results  are  likely  to  be  ob- 
tained. 

3.  A  normal  solution  of  sulphuric  acid. 

1  Zeitschr.  f.  Immunitatsforsch.,  1910,  vi,  16. 

2  Munch,  med.  Wochenschr.,  1910,  38,  1981. 

3  Deutsch.  med.  Wochenschr.,  1910,  50,  2333. 

4  Deutsch.  med.  Wochenschr.,  1911,  22,  1021. 
*  Jour.  Amer.  Med.  Assoc.,  1912,  lix,  1257. 

8  Berl.  klin.  Wochenschr.,  1911,  43,  1935. 


THE    RELATION    OF    LIPOIDS   TO    IMMUNITY  561 

4.  A  saturated  solution  of  barium  hydroxid  made  equivalent  to  the 
normal  solution  of  sulphuric  acid.     In  the  use  of  the  barium  hydroxid 
it  is  imperative  to  prevent  its  exposure  to  the  air.     A  solution  that  has 
become  cloudy,  owing  to  the  entrance  of  carbon  dioxid,  should  not  be 
used.     In  carrying  out  the  test  it  is  best  to  pour  out  the  amount  of  barium 
hydroxid  needed  for  the  test  into  a  rubber-stoppered  bottle  or  test-tube, 
so  as  not  to  contaminate  the  stock  solution. 

5.  A  1  per  cent,  alcoholic  solution  of  phenolphthalein  containing  1 
per  cent,  of  a  10  per  cent,  solution  of  strontium  chlorid.     The  strontium 
chlorid  has  been  found  to  catalyze  the  reaction. 

The  Test. — The  test  is  conducted  as  follows:  A  number  of  clean 
beakers  of  about  50  c.c.  capacity  are  used.  For  each  dilution  of  the 
serum  a  separate  beaker  is  required.  One  beaker  is  used  for  an  antigen 
control,  and  another  to  control  the  system  of  barium  hydroxid  and 
sulphuric  acid.  Five  beakers  may  be  used,  Nos.  1,  2,  and  3  constituting 
the  main  test,  No.  4  the  antigen  control,  and  No.  5  the  system  control. 

The  reagents  are  added  by  means  of  overflow  pipets.  To  each  of  the 
first  four  beakers  is  added  1  c.c.  of  the  dilute  antigen  to  be  used  in  the 
test  (about  1 :  10,000).  To  beaker  5  is  added  1  c.c.  of  the  salt  solution 
used  in  making  the  dilutions  of  the  antigen  and  the  serums.  Now  0.1 
c.c.  of  the  dilute  serum  to  be  tested  is  added  to  each  of  the  first  three 
beakers,  each  beaker,  however,  containing  the  same  serum  in  a  different 
dilution.  To  beaker  5  the  same  quantity  of  salt  solution  is  added,  but 
to  beaker  4 — the  antigen  control — no  serum  or  salt  solution  is  added. 

To  each  of  the  five  beakers  the  system  of  sulphuric  acid  and  barium 
hydroxid  and  phenolphthalein  is  now  added  carefully.  First,  2  c.c.  of 
the  normal  suphuric  acid  solution  are  added  to  each;  then  2  c.c.  of  the 
barium  hydroxid,  and  finally  0.1  c.c.  of  the  phenolphthalein  strontium 
chlorid  mixture. 

It  will  be  seen  that  beaker  4 — the  antigen  control — contains  all  the 
constituents  of  the  test-beakers  1  to  3  except  serum.  To  make  beaker 
4  qualitatively  as  well  as  quantitatively  equal  to  beakers  1  to  3,  0.1  c.c. 
of  the  dilute  serum  (the  average  of  the  dilutions  of  serum  which  are  used 
in  the  test)  is  now  added  to  beaker  4,  the  reaction  having  already  taken 
place. 

The  addition  of  the  sulphuric  acid  and  barium  hydroxid  requires 
great  care.  Since  the  reaction  depends  on  small  differences  in  acidity 
or  alkalinity,  it  is  obvious  that  slight  errors  will  vitiate  the  results.  For 
the  acid  and  the  alkali  separate  pipets  are  used.  After  emptying  the 

pipet  of  its  content  of  acid  or  alkali,  the  last  traces  adhering  to  the 
36 


562  THE    RELATION    OF    COLLOIDS    AND    LIPOIDS   TO    IMMUNITY 

inside  of  the  pipet  are  removed  by  washing.  These  washings  are  later 
added  to  the  beakers  to  which  they  belong.  In  filling  the  pipets.  with 
acid  or  alkali,  the  latter  should  first  be  drawn  up  into  the  pipet  at  least 
once,  and  then  emptied  again  before  the  pipet  is  finally  filled  for  delivery 
into  the  next  test-beaker.  Only  by  careful  attention  to  these  points 
in  the  technic  can  reliable  results  be  obtained. 

Reading  the  Results. — If  beakers  1  to  3  contained  the  antiserum  to  the 
antigen  used,  a  positive  epiphanin  reaction  will  be  obtained,  and  if  the 
barium  hydroxid  and  sulphuric  acid  were  previously  carefully  adjusted 
to  each  other,  it  will  be  found  that  beakers  1  to  3  will  be  lighter  than  the 
antigen  control,  beaker  4.  The  presence  of  a  specific  antigen-antibody 
combination  has  shifted  the  phenolphthalein  end-point  in  the  sense  of 
increased  H-ion  concentration.  The  exact  differences  in  the  alkalinity 
between  beakers  1  to  3  and  beaker  4  can  be  quantitatively  determined 
by  titration  with  JQQ  suphuric  acid,  and -the  results  expressed  as  a  curve. 

If  the  antigen  and  antibody  were  not  specific,  the  epiphanin  reac- 
tion will  be  negative.  Beakers  1  to  3  will  be  more  alkaline  than  the 
antigen  control,  beaker  4,  because,  as  previously  pointed  out,  serums 
alone  or  antigen  with  non-specific  serums  shift  the  phenolphthalein 
end-point  in  the  sense  of  increased  OH-ion  concentration. 

The  results  may  be  plotted  as  curves.  The  titration  values  in  JQQ 
sulphuric  acid  are  placed  on  the  ordinates,  and  the  serum  dilutions  on 
the  abscissae.  The  positive  values  are  plotted  above  the  line  and  the 
negative  values  below  the  line.  No  reaction  is  regarded  as  positive 
unless  it  gives  a  titration  value  of  at  least  0.05  c.c.  JQQ  sulphuric  acid. 
Values  below  0.05  c.c.  are  easily  within  the  limits  of  error. 

The  following  method,  employed  by  Seifert,  is  much  simpler,  but  is 
open  to  the  error  on  account  of  using  the  antigen  and  serum  in  too  con- 
centrated a  state.  In  a  small  test-tube  place  0.1  c.c.  of  a  1  :  10  solution 
of  the  serum  in  normal  salt  solution,  and  add  0.1  c.c.  of  an  alcoholic 
extract  of  syphilitic  liver.  To  this  slowly  add  1  c.c.  of  decinormal 
sulphuric  acid  and  1  c.c.  of  a  solution  of  barium  hydroxid  of  the  exact 
concentration  needed  to  neutralize  the  sulphuric  acid  solution.  On  the 
addition  of  the  drop  of  the  phenolphthalein  solution  the  fluid  turns  red 
when  the  serum  is  from  a  syphilitic,  whereas  no  change  in  tint  occurs 
with  non-syphilitic  serum. 

Practical  Value. — The  reaction  appears  to  be  of  considerable  value 
in  the  diagnosis  of  syphilis.  With  serums  and  antigen  in  proper  dilu- 
tions, the  results  closely  parallel  those  secured  by  the  Wassermann  reac- 


THE    RELATION    OF    LIPOIDS    TO    IMMUNITY  563 

tion.  Keidel  and  Kurwitz  report  positive  reactions  with  luetic  serums 
in  about  75  per  cent  of  their  cases.  The  reaction  was  found  highly 
specific  in  that  syphilitic  extracts  gave  negative  reactions  with  serums 
of  non-syphilitic  persons  and  patients  suffering  from  malignant  disease. 
Extracts  of  normal  fetal  liver  and  beef  heart  gave  negative  reactions 
with  serums  of  syphilitic  persons. 

Positive  reactions  have  also  been  found  in  malignant  disease,  as  with 
the  antigens  of  carcinoma  and  sarcoma.  Keidel  and  Hurwitz  obtained 
16  positive  reactions  in  a  series  of  24  serums  of  persons  suffering  with 
definite  or  suspected  malignant  disease.  Burmeister  did  not  find  the 
reaction  of  value  in  cancer. 

The  epiphanin  reaction  has  also  been  used  in  the  diagnosis  of  preg- 
nancy, but  sufficient  work  has  not  been  done  to  render  an  expression  as 
to  its  merits  of  value  at  this  time. 

THE  MIOSTAGMIN  REACTION 

Among  the  very  large  number  of  immunity  reactions  employed  in 
attempts  to  secure  a  diagnostic  test  for  cancer,  the  "miostagmin  reac- 
tion" of  Ascoli  and  Izar1  is  the  only  one  thus  far  devised  that  claims 
the  serious  attention  of  the  clinician. 

Principles. — This  reaction  is  founded  on  the  fact,  noted  by  Ascoli, 
that  by  the  mixing  of  an  antigen  and  its  corresponding  antibody  there 
results  a  reduction  of  the  surface  tension  of  the  liquid  containing  these, 
which  may  be  demonstrated  by  counting  the  number  of  drops  of  the 
fluid  in  a  given  volume  (usually  1  c.c.),  under  constant  conditions. 
Normal  serum  diluted  with  salt  solution  is  first  tested,  and  the  number  of 
drops  found  in  a  cubic  centimeter  determined  with  a  specially  devised 
instrument  known  as  Traube's  stalagmometer.  The  antigen  is  so  diluted 
that  when  mixed  with  this  normal  serum  it  does  not  increase  the  number 
of  drops  more  than  one  in  a  cubic  centimeter.  When  properly  diluted 
patient's  serum  and  antigen  are  mixed,  it  may  be  found  that  the  number 
of  drops  is  increased  from  2  to  8  in  a  cubic  centimeter.  This  constitutes  a 
positive  reaction.  The  reaction  is  apparently  due  to  the  lowering  of  sur- 
face tension,  so  that  more  and  smaller  drops  are  found;  hence  the  term 
"miostagmin"  has  been  applied  to  the  test,  the  word  being  devised  from 
the  Greek,  meaning  "small  drop." 

The  reaction  is  said  to  be  sharply  specific  and  very  delicate,  so  that 
antigens  diluted  up  to  1  :  100,000,000  or  higher  may  be  detected.  The 
technic  requires  considerable  practice  and  experience  or  erroneous 
results  are  quite  likely  to  occur. 

1  Miinch.  med.  Wochenschr.,  1910,  57,  62,  182,  and  403. 


564         THE    RELATION    OF    COLLOIDS    AND    LIPOIDS   TO    IMMUNITY 

The  exact  nature  of  the  reaction  is  not  known.  The  antigens  are 
soluble  in  alcohol,  but  their  nature  is  obscure.  The  antibody  involved 
in  the  reaction  is  referred  to  as  the  miostagmin,  but  its  relation  to  other 
antibodies  is  also  unknown.  It  is  probably  a  physicochemical  or  col- 
loidal reaction,  and  for  this  reason  it  has  been  placed  in  this  chapter. 

Technic. — The  antigen  is  most  difficult  to  prepare.  A  recent  method 
described  by  Ascoli  is  as  follows: 

1.  Cut  non-degenerated  portions  of  malignant  tumor   (cancer  or 
sarcoma)  into  small  pieces  and  dry  in  vacuo  or  spread  out  in  a  thin 
layer  on  clean  glass  plates  and  keep  at  a  temperature  of  37°  C. 

2.  Pulverize  the  dried  substance  and  extract  with  pure  methyl 
alcohol  (in  the  proportion  of  5  gm.  to  25  c.c.)  for  twenty-four  hours  at 
50°  C.  in  closed  vessels,  and  shake  occasionally. 

3.  Filter  while  still  hot,  and  allow  the  filtrate  to  cool,  and  then  filter 
again  through  Schleicher  and  Schull's  filter-paper  No.  590. 

4.  It  is  now  necessary  to  titrate  the  antigen  and  to  determine  in  what 
dilution  it  should  be  employed.     Various  dilutions  of  the  antigen  are 
made  with  distilled  water,  as,  e.  g.}  1  :  10,  1  :  25,  1  :  50,  1  :  100,  1  :  150, 
1 : 200,  etc.     A  fresh  normal  serum  is  diluted  1  :  20  with  normal  salt 
solution,  and  9  c.c.  of  this  are  mixed  with  1  c.c.  of  the  various  antigen 
dilution.     Into  another  tube  place  9  c.c.  of  the  diluted  serum  and  1  c.c. 
of  distilled  water.     All  test-tubes,  pipets,  and  other  glassware  used  must 
be  perfectly  dry. 

The  tubes  are  gently  shaken  and  placed  in  an  incubator  at  37°  C. 
for  two  hours.  The  drop  number  for  each  fluid  is  then  estimated  by 
Traube's  stalagmometer.  This  instrument  is  merely  a  finely  and 
elaborately  graduated  pipet  with  a  central  bulbous  reservoir.  The 
dropping  end  of  the  instrument  ends  in  a  flattened  ground  base,  thus 
insuring  uniformity  in  the  size  of  the  drops.  The  instrument  is  so 
graduated  that  a  fraction  of  a  drop  can  be  estimated.  That  antigen  is 
to  be  chosen  that  does  not  alter  the  drop  number  for  normal  serum  by  more 
than  one  drop  in  a  cubic  centimeter — the  strongest  dilution  that  fulfils  this 
condition  being  chosen. 

The  Test. — The  patient's  serum  is  diluted  1  : 20  with  normal  salt 
solution  and  its  drop  number  determined.  Then  take  two  tubes,  and 
into  one  place  9  c.c.  of  diluted  serum  plus  1  c.c.  of  antigen  dilution; 
into  the  other  place  9  c.c.  of  diluted  serum  plus  1  c.c.  of  distilled  water. 
A  third  tube  may  be  prepared,  which  should  contain  9  c.c.  of  normal 
serum  (1  :  20)  plus  1  c.c.  of  the  same  antigen  dilution.  A  fourth  tube 
contains  9  c.c.  of  a  known  positive  serum  (1 :  20)  from  a  case  of  cancer 
and  1  c.c.  of  the  antigen  dilution. 


THE    RELATION    OF    LIPOIDS    TO    IMMUNITY 


565 


All  tubes  should  be  carefully  labeled,  their  drop  numbers  determined, 
and  then  placed  in  an  incubator  at  37°  C.  for  two  hours  or  in  the  water- 
bath  at  50°  C.  for  one  hour.  At  the  end  of  this  time  they  are  removed, 
allowed  to  cool,  and  the  drop  number  of  each  is  determined. 

The  controls  are  first  examined  to  show  that  the  antigen  has  not 
undergone  any  change.  Variations  of  the  number  above  one  and  a  half 
or  two  drops  (as  compared  with  the  control  containing  distilled  water  in- 
stead of  antigen)  .are  regarded  as  positive  reactions.  The  increase  in  drops 
is  seldom  greater  than  eight. 

TABLE  21.— MIOSTAGMIN  REACTION  IN  CANCER 


DROPS  PER  C.c. 

DROPS  PER  C.c. 

CONTROLS  9  C.c. 

9  C.c.  SERUM  (1:20)  PLUS  1  C.c. 
ANTIGEN  (1:200) 

AFTER  INCUBA- 
TION AT  37°  C. 

SERUM  (1:20)  PLUS 
1  C.c.  DISTILLED 

RESULTS 

FOR  Two  HOURS 

WATER  AFTER 

INCUBATION 

Normal  serum  .  .    .          

56.0 

56.4 

Negative 

Known  cancer  serum  

62.4 

59.2 

Positive 

Unknown  serum  for  diagnosis  

612 

60.0 

Positive 

Unknown  serum  for  diagnosis  
Unknown  serum  for  diagnosis  . 

57.2 
62.4 

57.0 
59.8 

Negative 
Positive 

Other  Methods  for  Preparing  Antigens. — Various  methods  for  pre- 
paring antigen  and  conducting  the  test  are  to  be  found  in  the  literature, 
and  it  is  extremely  difficult  to  arrive  at  a  correct  conclusion  as  to  which 
is  the  best  method  for  preparing  antigen.  Among  these  methods  for 
the  preparation  of  antigen  other  than  those  previously  described  are 
the  following: 

1.  After  securing  the  alcoholic  extract  described  elsewhere  evaporate 
it  to  dryness.     Again  extract  in  methyl  alcohol  and  evaporate.     Extract 
with  warm  ether,  renewed  several  times  during  the  course  of  twenty- 
four  hours.     Dry,  and  repeat  the  extraction  several  times  until  the 
alcohol  remains  colorless.     Evaporate  the  alcoholic  and  ethereal  ex- 
tracts at  50°  and  37°  C.  respectively.     A  yellowish-red,  sticky  mass 
results.     Dissolve  this  in  a  large  amount  of  water-free  ether.     Filter, 
and  evaporate  at  room  temperature  until  a  slight  powdery  precipitate 
is  deposited.     This  solution  constitutes  the  stock  antigen,  which,  how- 
ever, may  require  still  further  concentration. 

2.  A  synthetic  cancer  antigen  may  be  prepared  by  grinding  up  0.5 
gram  of  lecithin  (ovolecithin  Merck,  or  lecithin  Richter),  and  extract  it 
with  50  c.c.  of  acetone  for  twenty-four  hours  at  50°  C.     Filter  through 
Scheicher  and  Schull's  filter-paper  No.  590,  until  clear.     Just  before 


566         THE    KELATION    OF    COLLOIDS   AND    LIPOIDS   TO    IMMUNITY 

it  is  to  be  used  it  should  be  dilated  with  water  in  such  amount  that  1  c.c. 
will  contain  the  largest  amount  that  does  not  cause  a  marked  reduction 
of  surface  tension  in  normal  serum.  As  a  rule,  this  dilution  is  between 
1  :  50  and  1 : 100  (Kohler  and  Luger)1. 

3.  A  syphilis  antigen  may  be  prepared  by  extracting  0.5  gram  of 
dried  and  powdered  syphilitic  liver  with  50  c.c.  of  absolute  alcohol  for 
two  hours  at  37°  C.  with  frequent  shaking.     Filter,  and  concentrate  to 
10  c.c. 

4.  A  bacterial  antigen,  as  e.  g.,  one  of  typhoid  bacilli,  may  be  prepared 
as  follows :  Wash  off  five  forty-eight-hour  agar  cultures  of  typhoid  bacilli 
with  5  c.c.  of  normal  salt  solution  for  each  tube.     Cover  the  emulsion 
with  toluol,  and  shake  vigorously  for  several  hours.     Place  in  an  in- 
cubator at  37°  Cf  for  forty-eight  hours,  and  filter  through  a  sterile 
Berkefeld  filter.     This  filtrate  may  be  used  as  antigen,  or  it  may  be 
used  in  preparing  an  alcoholic  extract  in  the  following  manner:   To  the 
original  aqueous  filtrate  add  50  c.c.  of  absolute  alcohol.     Allow  the 
mixture  to  stand  for  one-half  hour,  shake,  centrifugate,  and  then  mix 
the  sediment  with  20  c.c.  of  absolute  alcohol.     Shake  thoroughly  once 
more,  and  again  centrifugate.     Combine  the  two  extracts,  and  con- 
centrate on  the  water-bath  to  about  20  c.c. 

Practical  Value. — This  test  is  quite  delicate,  and  errors  due  to  faulty 
technic  are  quite  likely  to  creep  in.  Unless  all  precautions  are  rigidly 
observed,  the  results  are  worthless.  Although  an  extensive  literature 
has  accumulated  bearing  evidence  as  to  the  value  of  the  test  as  a  diag- 
nostic procedure,  the  method  has  not,  however,  come  into  general  use. 

Ascoli  and  Izar  especially  have  advocated  the  test  in  the  diagnosis  of 
cancer.  In  100  cases  of  malignant  tumors,  they  obtained  93  positive 
reactions;  in  103  cases  of  other  diseases  they  obtained  only  one  positive 
reaction.  Tedesko,  Stabilini,  Leitch,  Kelling,  and  others  have  reported 
favorably  upon  the  practical  value  of  the  test  in  the  diagnosis  of  cancer. 
Burmeister2  has  found  that  a  negative  reaction  has  some  value  in 
excluding  cancer,  and  is  of  more  value  in  arriving  at  a  diagnosis  than  a 
positive  reaction,  i.  e.}  it  has  a  higher  negative  than  a  positive  value. 

The  test  has  also  been  used  in  the  diagnosis  of  typhoid  fever,  para- 
typhoid fever,  syphilis,  tuberculosis  (positive  only  in  active  cases) 
echinococcus  disease,  etc.  Obviously,  other  methods  of  diagnosis,  such 
as  the  agglutination  reaction  and  the  Wassermann  reaction,  have  super- 
seded this  test  in  practical  diagnosis.  The  method  possesses,  however, 
considerable  theoretic  interest  and  is  worthy  of  further  investigation. 

1  Wien.  klin.  Wochenschr.,  1912,  25, 1114.        2  Jour.  Infect.  Dis.,  1913, 12,  459. 


CHAPTER  ^XXVII 
ANAPHYLAXIS 

IT  is  generally  believed  that  when  an  animal  previously  injected  with 
an  antigenic  substance  is  subsequently  reinjected  with  the  same  sub- 
stance, the  antibodies  induced  by  the  first  injection  are  reenforced,  and 
that  a  continuation  of  the  process  of  immunization  will  eventually  lead  to 
a  high  degree  of  immunity.  Under  certain  circumstances,  however,  this 
is  not  the  case,  because  severe  and  even  fatal  symptoms,  as  well  as  other 
manifestations,  may  set  in  after  the  second  injection,  indicating  that, 
instead  of  being  immune,  the  animal  is  indeed  hypersusceptible  or 
hypersensitive  to  the  affects  of  the  antigenic  substance. 

Similarly,  it  is  a  common  observation  that  whereas  certain  infections, 
such  as  smallpox,  scarlet  fever,  and  measles,  confer  a  state  of  immunity, 
others,  as,  for  example,  pneumonia,  erysipelas,  and  influenza,  not  only 
are  not  followed  by  immunity,  but,  indeed,  that  a  decreased  resistance 
or  predisposition  to  subsequent  infection  by  the  same  microorganism 
may  be  induced. 

Before  experimental  investigation  of  this  subject  was  undertaken, 
not  a  few  observations  were  made  and  described  by  the  early  workers 
in  the  fields  of  bacteriology  and  immunity  that  correspond  exactly  with 
the  phenomenon  of  hyper  sensitiveness,  as  we  understand  it  today, 
although  the  true  explanation  of  their  unexpected  results  was  not  sus- 
pected, and  they  were  modestly  ascribed  to  faulty  technic,  embolism, 
toxicity  of  the  inoculum,  etc.  From  this  it  followed  that  the  discovery 
that  the  experimental  injection  of  such  ordinary  innocuous  substances 
as  normal  serum  and  milk  may  produce  violent  symptoms  and  death 
gave  rise  to  much  surprise  and  incredulity,  since  scientists  had  long  been 
accustomed  to  regard  the  reaction  of  an  animal  to  an  injection  as  a 
process  of  immunization,  or  diminished  sensitiveness,  instead  of  one  of 
increased  sensitiveness.  Here,  as  Besredka  remarked,  the  rules  of 
immunity  are  "  standing  on  their  heads." 

To  this  state  of  hypersensitiveness  Richet,  one  of  the  earliest  in- 
vestigators in  this  field,  applied  the  term  "  anaphylaxis,"  meaning 
"without  protection."  While  it  appears  to  be  the  exact  antithesis  of 
"immunity,"  which  means  "with  resistance  to  infection,"  recent  re- 

567 


568  ANAPHYLAXTS 

searches  would  tend  to  indicate  that  the  two  subjects  are  indeed  closely 
related.  An  enormous  amount  of  work  has  already  been  done  on  ana- 
phylaxis,  the  subject  being  of  the  greatest  importance,  not  only  on  ac- 
count of  its  practical  bearing  on  serum  therapy,  but  because  of  its  inti- 
mate relationship  to  the  subjects  of  infection  and  immunity,  and  the 
new  light  that  these  studies  may  throw  upon  the  nature  and  mechanism 
of  these  processes.  Although  the  phenomena  of  anaphylaxis  are  now 
known  to  be  due  to  the  proteins,  and  while  the  symptoms  and  lesions 
of  the  condition  are  fairly  well  understood,  the  exact  nature  and  mecha- 
nism involved  in  the  process  are  not  established,  and  the  entire  subject 
is  fraught  with  so  much  interest  as  regards  infection  and  immunity  that 
it  affords  a  fruitful  field  for  further  research. 

In  this  chapter  will  be  presented  the  known  facts  regarding  anaphy- 
laxis and  the  theories  that  have  been  advanced  in  explanation  of  its 
nature  and  mechanism,  the  consideration  of  anaphylaxis  in  its  practical 
application  to  medicine  being  left  for  the  following  chapter. 

Historic. — The  first  observation  of  anaphylaxis  as  it  occurs  in  an 
infectious  disease  was  probably  made  by  Jenner  in  1798.  This  investi- 
gator observed  the  sudden  appearance  of  an  "efflorescence  of  a  palish 
red  color"  about  the  parts  where  variolous  matter  had  been  injected  into 
a  woman  who  had  had  cowpox  thirty-one  years  before. 

In  1839  Magendi  found  that  rabbits  that  had  been  injected  with  egg- 
albumin  died  after  a  repetition  of  the  injection,  a  phenomenon  strikingly 
similar  to  that  observed  sixty-five  years  later  by  Theobald  Smith 
following  injections  of  horse  serum.  This  phenomenon  was  subsequently 
studied  thoroughly  by  Rosenau  and  Anderson  and  Otto. 

While  the  effects  of  diphtheria  and  tetanus  antitoxins  were  being 
studied,  peculiar  and  apparently  paradoxic  results  were  occasionally 
observed  during  immunization  of  animals  with  the  bacterial  toxins. 
Thus  in  1895  Brieger l  reported  the  case  of  a  goat  that  was  highly  im- 
munized against  tetanus  and  yet  was  subject  to  tetanus.  In  1901  von 
Behring  and  Kitashima2  reported  similar  findings  with  diphtheria  in 
horse  immunized  against  that  infection.  At  this  time  it  was  shown  that 
the  results  could  not  be  due  to  the  cumulative  effect  of  the  toxin,  and 
the  explanation  offered  aimed  to  show  that  the  process  was  purely  his- 
togenetic,  and  based  upon  the  assumption  that  receptors  attached  to  the 
body-cells  had  a  closer  affinity  for  toxin  than  the  free  (antitoxin)  re- 
ceptors in  the  blood-stream.  At  the  present  time  toxin  hypersuscep- 
tibility  is  held  by  some  to  be  a  true  anaphylactic  reaction  brought  about 

1  Zeitschr.  f.  Hyg.,  1895,  101.  2  Berl.  klin.  WochenscKr.,  1901,  xxxviii,  157. 


VON  PIRQUET'S  EARLY  STUDIES  569 

by  protein  substances  in  the  toxin  filtrate;  others  regard  von  Behring's 
explanation  as  satisfactory.  Further  reference  to  this  subject  will  be 
made  later  on  in  this  chapter. 

Richet's  Studies. — The  fundamental  observations  upon  which  our 
present  knowledge  of  anaphylaxis  is  based  were  made  in  1898  by 
Hericourt  and  Richet.1  These  observers  found  that  repeated  injections 
of  eel  serum  into  dogs  gave  rise  to  an  increased  susceptibility  to  this 
substance,  instead  of  immunizing  the  dogs  against  the  serum.  These 
studies  were  continued  by  Richet 2  and  his  assistants  with  extracts  of  the 
tentacles  of  certain  sea  anemones.  These  studies  showed  that  a  second 
injection  of  the  poison  into  dogs,  given  after  an  interval  of  several  days, 
is  followed  by  greater  and  more  intense  activity  than  marked  the  first 
injection.  If  the  animal  survives,  however,  the  disease  is  conquered 
more  readily  after  the  second  than  after  the  first  injection.  As  pre- 
viously stated,  Richet  coined  the  word  "anaphylaxis,"  meaning  "with- 
out protection,"  and  indicating  that  the  first  injection  destroyed  any 
natural  resistance  that  the  animal  might  possess  against  the  poison 
(actionocongestin).  From  these  studies  he  concluded  that  two  dif- 
ferent substances  are  contained  in  eel  serum  and  in  the  tentacles  of 
actiniens,  one  concerned  in  establishing  an  immunity,  and  the  other  in 
calling  forth  a  hypersensitiveness;  thus  far,  however,  the  separate 
existence  of  these  two  hypothetic  substances  has  not  been  proved. 

Arthus  Phenomenon. — In  1903  Arthus,3  at  the  instigation  of  Richet, 
showed  that  similar  results  may  be  obtained  with  non-toxic  substances, 
like  serum  and  milk.  On  injecting  rabbits  at  definite  intervals  with 
normal  horse  serum,  he  found  that  the  first  two  or  three  doses  were 
absorbed,  whereas  subsequent  injections,  given  subcutaneously,  led  to 
increasingly  severe  local  reactions  (Arthus  phenomenon) .  If  the  animals, 
however,  were  first  injected  subcutaneously  and  later  intravenously, 
or  intraperitoneally,  serious  symptoms  of  dyspnea,  convulsions,  and 
diarrhea,  and  even  death  resulted. 

Von  Pirquet's  Early  Studies. — For  a  long  time  urticarial  eruptions 
were  occasionally  observed  to  follow  transfusion  of  blood  from  lambs 
and  other  lower  animals  to  persons  suffering  from  anemia  and  similar 
conditions.  Soon  after  diphtheria  antitoxin  was  discovered  the  medical 

1  Compt.  rend.  Soc.  de  Biol.,  1898,  53. 

2  Compt.  rend.  Soc.  de  Biol.,  1902,  liv,  170;   1903,  Iv,  246;   1904,  Ivi,  302;   1905, 
Iviii,  112;    1907,  Ixii,  358,  643;    1909,  Ixvi,  763;    1909,  Ixvi,  810;    1909,  Ixvi,  1005. 
Ann.  de  1'Inst.  Pasteur,  1907,  xxi,  497,  1908,  xxii,  465. 

3  Compt.  rend.  Soc.  de  Biol.  1903,  Iv,  817;    1906,  Ix,  1143.     Archiv.  internat.  de 
Physiol.,  1908-09,  vii,  472. 


570  ANAPHYLAXIS 

profession  was  shocked  to  learn  of  the  sudden  death  of  the  healthy  child 
of  an  eminent  German  professor  following  a  prophylactic  injection  of  the 
serum.  In  1902  von  Pirquet  began  the  study  of  these  clinical  mani- 
festations with  a  child  in  Escherich's  clinic  who,  after  receiving  a  second 
dose  of  horse  serum  ten  days  after  the  first,  on  the  same  day  developed 
symptoms  of  fever  and  a  rash.  On  the  basis  of  this  observation  von 
Pirquet 1  reached  the  conclusion  that  the  prevailing  views  regarding  the 
length  of  the  incubation  period  of  an  infectious  disease  could  not  be 
correct.  He  therefore  propounded  the  theory  that  the  organism  con- 
cerned in  the  etiology  of  disease  calls  forth  symptoms  only  when  it  has 
been  altered  by  antibodies,  the  period  of  incubation  representing  the 
interval  necessary  for  the  formation  of  these  antibodies. 

In  conjunction  with  Schick,  von  Pirquet  endeavored  to  study  all 
infectious  diseases  from  the  same  point  of  view,  especially  smallpox, 
measles,  recurrent  fever,  streptococcus  infections,  and  the  reactions  to 
cowpox  virus,  tuberculin,  and  mallein.  Later  these  same  observers2 
studied  the  symptoms  following  injection  and  reinjection  of  horse  serum, 
designating  the  train  of  symptoms  "serum  sickness."  They  empha- 
sized that  a  single  injection  of  serum  may  suffice  to  bring  about  the 
symptoms,  and  that  this  immediate  reactivity  possesses  diagnostic 
value  in  so  far  as  it  enables  us  to  decide  whether  a  previous  infection 
has  occurred.  How  near  the  astute  Jenner  came  to  reaching  the  same 
conclusion  is  shown  in  the  following  abstract  from  his  report  in  1798: 

"  It  is  remarkable  that  variolous  matter,  when  the  system  is  disposed 
to  reject  it,  should  excite  inflammation  on  the  part  to  which  it  is  applied 
more  speedily  than  when  it  produces  the  smallpox.  Indeed,  it  becomes 
almost  a  criterion  by  which  we  can  determine  whether  the  infection  will  be 
received  or  not  (italics  ours).  It  seems  as  if  a  change,  which  endures 
through  life,  had  been  produced  in  the  action,  or  disposition  to  action,  in 
the  vessels  of  the  skin;  and  it  is  remarkable,  too,  that  whether  this 
change  has  been  effected  by  the  smallpox  or  the  cowpox,  that  the  dispo- 
sition to  sudden  cuticular  inflammation  is  the  same  on  the  application 
of  variolous  matter." 

Von  Pirquet  at  this  time  proposed  the  term  "allergy,"  from  ergeia, 
reactivity,  and  allos,  altered,  meaning  altered  energy  or  a  changed  re- 
activity, as  a  clinical  conception  expressing  a  truth  without  binding 

1  Zur  Theorie  der  Infectionskrankheiten  (Vorlaufige  Mitteilung),  April  2,  1903. 
"Zur  Theorie  der  Vaksination, "  Verhandl.  d.  Gesellsch.  f.  Kinderh.,  Kassel,  1903. 

2  Wien.  klin.  Wochenschr.,  1903,  xvi,  758,  1244;    1905,  xviii,  531.     Die  Serum- 
krankheit,  Leipsic,  Deuticke,  1905;    Munch,  med.  Wochenschr.,  1906,  liii,  66.     For 
a  full  bibliography  on  this  subject  of  allergy  up  to  1910  see  von  Pirquet,  Archiv.  Int. 
Med.,  1911,  vii,  259  and  383. 


DEFINITION  571 

any  one  to  a  theory  based  upon  bacteriologic,  pathologic,  or  biologic 
findings. 

Theobald  Smith  Phenomenon. — While  von  Pirquet  was  making 
these  studies,  great  impetus  was  given  the  experimental  study  of  ana- 
phylaxis  by  the  observation  of  Theobald  Smith,  who  found  that  guinea- 
pigs  that  were  used  for  standardizing  the  strength  of  diphtheria  anti- 
toxin after  a  second  injection  of  serum  frequently  presented  symptoms 
of  a  serious  character,  such  as  great  restlessness,  dyspnea,  itching  of  the 
skin,  and  violent  convulsive  seizures.  In  fully  50  per  cent  of  the  animals 
death  occurred  within  half  an  hour. 

Simultaneously  Rosenau  and  Anderson1  in  this  country  and  Otto2 
in  Germany  undertook  the  study  of  this  phenomenon.  The  first-named 
investigators  showed  most  conclusively,  by  a  thorough  series  of  experi- 
ments, the  action  of  horse  serum  and  other  substances  in  guinea-pigs, 
and  proved  that  serum  sickness  was  due  to  some  constituent  of  the 
serum  independent  of  the  antitoxic  antibodies,  as  normal  horse  serum 
yielded  exactly  similar  results. 

Among  the  earlier  studies  of  anaphylaxis  of  importance  were  those 
of  Weichardt.3  These  were  made  with  extracts  of  placental  cells,  and 
later  with  the  proteins  of  pollen,  in  relation  to  hay-fever.  Wolff-Eisner4 
wrote  a  treatise  that  had  as  its  fundamental  idea  the  belief  that  hyper- 
sensibility  was  due  to  endotoxins  liberated  by  a  lysin  formed  as  a  result 
of  the  first  injection.  Also  among  the  earliest  and  most  valuable  studies 
upon  the  nature  of  anaphylaxis,  and  showing  the  important  relation  of 
proteins  to  the  process,  are  those  of  Vaughan5  and  his  coworkers;  indeed 
the  studies  of  Smith,  Rosenau  and  Anderson,  Vaughan  and  Wheeler, 
Gay  and  Southard,  Auer  and  Lewis,  and  others  have  gained  for  America 
a  prominent  part  in  the  development  of  this  important  subject. 

Definition. — By  anaphylaxis,  in  the  limited  meaning  of  the  term,  as, 
e.  g.,  in  that  following  the  injection  of  horse  serum  in  man  or  following 
the  experimental  administration  of  practically  any  protein  in  the  lower 
animals,  is  understood  the  following  train  of  phenomena.  When  a 
foreign  protein  is  introduced  into  the  animal  body,  usually  parenterally, 

1  Bull.  29,  Hyg.  Lab.,  U.  S.  P.  H.  and  M.  H.  S.,  1906;  Bull.  36,  Hyg.  Lab.,  April, 
1907;   Jour.  Infect.  Dis.,  1907,  iv,  552;    Bull.  45,  Hyg.  Lab.,  June,  1908;    Bull.  50, 
Hyg.  Lab.,  1909;    Jour.  Amer.  Med.  Assoc.,  1906,  xlvii,  1007;    Archiv.  Int.  Med., 
1909,  iii,  519. 

2  von  Leuthold  Gedenkschrift,  1905,  i;    Munch,  med.  Wochenschr.,  1907,  liv, 
1665;   Kolle  and  Wassermann,  1908,  ii,  255. 

3  Berl.  klin.  Wochenschr.,  1903,  No.  1. 

4  Zeitschr.  f.  Bakteriol.,  1904;    Berl.  klin.  Wochenschr.,  1904,  xli,  1105,  1131, 
1156,  1273;   Munch,  med.  Wochenschr.,  1906,  liii,  217. 

5  Summarized  in  "Protein  Split  Products,"  Lea  and  Febiger,  1913. 


572  ANAPHYLAXIS 

after  a  time  a  specific  hypersensitiveness  of  the  animal  for  this  pro- 
tein will  appear.  After  a  definite  interval,  a  second  injection  of  the 
same  substance,  harmless  in  itself,  may  produce  an  itching  rash  and 
fever  or  violent  symptoms  of  illness,  and  rapid  death  may  even  occur 
in  an  animal  so  inoculated.  In  other  words,  the  first  injection  of  the 
protein  (serum,  milk,  egg-albumen,  etc.)  produces  no  symptoms,  but 
serves  to  alter  the  power  of  reaction  on  the  part  of  the  body  cells  by  rendering 
them  unusually  sensitive  or  susceptible  to  the  same  or  to  closely  related 
foreign  protein.  Therefore,  as  defined  by  Rosenau,  anaphylaxis  may  be 
considered  as  "a  condition  of  unusual  or  exaggerated  susceptibility  of  the 
organism  to  foreign  proteins." 

Terminology. — As  previously  stated,  the  word  "anaphylaxis "(ana, 
against,  and  phylax,  guard,  or  phylaxis,  protection)  was  given  to  the 
condition  by  Richet,  because  he  considered  it  one  "  without  protection," 
or  a  state  just  the  opposite  to  immunity,  or  prophylaxis.  In  the  sense 
in  which  the  phenomenon  is  now  regarded  the  word  is  a  misnomer,  for 
we  look  upon  the  condition  of  hypersusceptibility  as  a  step  toward  the 
attainment  of  a  state  of  immunity,  and  as  a  distinct  benefit  and  ad- 
vantage to  the  organism.  The  term  " allergy,"  introduced  by  von 
Pirquet,  is  more  appropriate,  as  it  expresses  the  condition  of  the  body- 
cells,  i.  e.}  their  hypersensitiveness  or  altered  reactivity,  regardless  of 
any  theories  we  may  entertain  as  to  the  manner  in  which  this  change  is 
brought  about  or  manifested.  The  word  anaphylaxis  has,  however,  come 
into  general  use,  and  with  this  explanation,  we  may  continue  to  so  use  it. 

The  term  anaphylactogen  is  applied  to  the  protein,  as  serum,  milk, 
egg-albumen,  etc.,  which  sensitizes  the  body-cells;  sentizer  is  also  a  good 
word,  and  the  process  of  rendering  body-cells  hypersensitive  by  ad- 
ministering a  foreign  protein  has  been  called  sensitization.  In  von 
Pirquet's  nomenclature  the  protein  would  be  called  an  allergen. 

The  term  anaphylatoxin  is  applied  to  the  toxic  substance  believed  to 
be  formed  at  the  time  of  reinjection  of  the  protein,  and  is  regarded  as 
responsible  for  the  lesions  and  symptoms  of  anaphylaxis.  In  the  belief 
that  anaphylaxis  resembles  an  intoxication,  the  second  inoculation  of 
protein  is  frequently  spoken  of  as  the  intoxicating  dose. 


PHENOMENA  OF  ANAPHYLAXIS 

The  essential  symptoms  and  lesions  of  anaphylaxis  vary  in  the  dif- 
ferent animals,  and,  indeed,  they  have  been  found  to  vary  among  animals 
of  the  same  species  under  different  experimental  conditions. 


PHENOMENA   OF  ANAPHYLAXIS  573 

Man. — When  it  is  remembered  that  anaphylaxis  and  immunity  are 
closely  interwoven,  and  that  anaphylaxis  may  be  but  a  step  toward 
securing  prophylaxis  and  immunity,  it  will  readily  be  understood  that 
under  the  varying  conditions  of  different  injections  the  phenomena  may 
be  quite  dissimilar.  One  of  the  best  known  examples  of  a  general 
anaphylactic  phenomenon  in  man  is  that  following  the  injection  of  a 
foreign  serum,  as,  e.  g.,  horse  serum  (diphtheria  antitoxin),  which  is 
characterized  by  an  itching  urticarial  eruption,  fever,  and  joint  pains, 
and  which  is  commonly  known  as  "serum  sickness."  Fortunately,  the 
severer  and  fatal  forms  of  anaphylaxis  in  man  are  extremely  rare,  most 
cases  having  occurred  in  persons  known  to  be  hypersensitive  to  horse 
protein  or  in  those  suffering  from  the  condition  known  as  status  lym- 
phaticus.  Familiar  local  anaphylactic  reactions  are  the  tuberculin, 
mallein,  and  luetin  reactions.  With  this  brief  statement  we  shall  pass 
to  a  consideration  of  anaphylaxis  in  the  lower  animals,  experimentation 
having  given  us  some  insight  into  the  mechanism  of  the  process.  Ana- 
phylaxis in  man  and  the  relation  it  bears  to  immunity  and  disease,  will 
be  discussed  again  in  Chapter  XXVIII. 

Guinea-pig. — This  animal  gives  the  most  constant  and  the  most 
intense  symptoms.  According  to  Doerrr,  guinea-pigs  are  four  hundred 
times  as  sensitive  an  anaphylactic  reagent  as  the  rabbit. 

Horse  serum,  when  injected  into  normal  guinea-pigs,  gives  rise  to 
no  symptoms.  As  much  as  20  c.c.  may  be  injected  into  the  peritoneal 
cavity,  and  small  amounts  may  even  be  injected  into  the  brain  without 
causing  any  untoward  symptoms. 

When  a  small  dose  of  serum  is  injected  intravenously,  intraperito- 
neally,  or  subcutaneously,  and  ten  days  later  a  second  injection  is  made, 
the  animal  develops  symptoms  of  acute  anaphylactic  asphyxia,  which, 
in  the  majority  of  instances,  terminates  fatally.  "  In  five  or  ten  minutes 
after  injection  the  pig  becomes  restless  and  then  manifests  indications 
of  respiratory  embarrassment  by  scratching  at  the  mouth,  coughing, 
and  sometimes  of  spasmodic,  rapid,  or  irregular  breathing;  the  pig 
becomes  agitated,  and  there  is  a  discharge  of  urine  and  feces.  This 
stage  of  exhilaration  is  soon  followed  by  one  of  paresis  or  complete 
paralysis,  with  arrest  of  breathing.  The  pig  is  unable  to  stand,  or  if  it 
attempts  to  move,  falls  upon  its  side;  when  taken  up  it  is  limp;  spas- 
modic, jerky  and  convulsive  movements  now  supervene.  This  chain  of 
symptoms  is  very  characteristic,  although  they  do  not  always  follow  in 
the  order  given.  Pigs  in  the  state  of  complete  paralysis  may  fully 
recover,  but  usually  convulsions  appear,  and  are  almost  invariably  a 


574  ANAPHYLAXIS 

forerunner  of  death.  Symptoms  appear  about  ten  minutes  after  the 
injection  has  been  given;  occasionally  in  pigs  not  very  susceptible  they 
are  delayed  thirty  to  forty-five  minutes.  Animals  developing  late 
symptoms  are  not  very  susceptible  and  do  not  die.  Death  usually 
occurs  within  an  hour,  and  frequently  in  less  than  thirty  minutes.  If 
the  second  injection  be  made  directly  into  the  brain  or  circulation,  the 
symptoms  are  manifested  with  explosive  violence,  the  animal  frequently 
dying  within  two  or  three  minutes"  (Rosenau). 

H.  Pfeiffer  has  shown  that  a  depression  of  the  temperature  is  a  con- 
stant finding  in  the  severer  forms  of  anaphylaxis  in  the  guinea-pig.  In 
fatal  cases  this  decrease  may  be  as  much  as  from  7°  to  13°  C.  Some 
relation  exists  between  the  extent  and  the  duration  of  the  fall  of  temper- 
ature and  the  severity  of  the  symptoms.  During  acute  anaphylaxis  the 
blood  shows  a  leukopenia, — a  diminution  in  complement, — and,  as  shown 
by  Friedberger,1  a  delay  in  or  a  loss  of  coagulability.  The  most  striking 
change  observed  after  death  is  permanent  distention  of  the  lungs,  resem- 
bling emphysema,  described  by  Gay  and  Southard,  and  particularly  by 
Auer  and  Lewis.2  The  lungs  do  not  collapse,  but  remain  fully  distended, 
forming  a  cast  of  the  pleural  cavities.  The  alveoli  are  distended,  and 
in  some  instances  the  walls  may  be  ruptured.  The  walls  of  the  second- 
ary and  tertiary  bronchi  are  contracted,  with  infoldings  of  the  normally 
thick  mucosa,  due  to  contraction  of  the  smooth  muscle  by  peripheral 
action,  death  really  resulting  from  inspiratory  immobilization  of  the 
lungs.  The  heart  continues  to  beat  long  after  respiration  has  ceased. 
Rosenau3  and  Gay  and  Southard4  have  also  described  minute  hemor- 
rhages in  various  organs  and  mucous  membranes. 

The  amount  of  serum  necessary  to  sensitize  a  guinea-pig  is  sur- 
prisingly small.  Rosenau  and  Anderson  found  one  guinea-pig  that  was 
sensitized  by  0.000,001  c.c.  As  a  rule  they  used  less  than  0.004  c.c.  in 
their  experiments.  Besredka  places  the  minimum  amount  necessary 
to  secure  uniform  results  at  0.001  c.c.,  whereas  0.0001  c.c.  proved  suf- 
ficient in  a  considerable  percentage  of  animals.  The  sensitizing  dose  of 
horse  serum  ordinarily  employed  in  experiments  upon  guinea-pigs  is 
0.01  c.c. ;  amounts  ranging  from  0.001  c.c.  to  1  c.c.  are  ordinarily  followed 
by  an  incubation  period  of  from  ten  to  sixteen  days.  Large  doses  also 
sensitize,  but  a  longer  incubation  period  is  required.  In  order  to  pro- 
duce a  fatal  result,  the  second  or  intoxicating  dose  must  be  considerably 

1  Zeitschr.  f.  Immunitatsf.,  1  orig.,  1909-1910,  8,  636. 

2  Jour.  Exp.  Med.,  1910,  xii.  172.  3  Bull.  No.  32  of  the  Hyg.  Lab.,  1906. 
4  Jour.  Med.  Research,  1908,  xix,  1,  5,  17. 


PHENOMENA  OF  ANAPHYLAXIS  575 

larger  than  the  minimum  sensitizing  dose,  the  proportion  between  the 
two  having  been  placed  by  Doerr  and  Russ  as  1  :  1000,  i.  e.}  if  0.001  c.c. 
of  serum  is  injected  intraperitoneally  in  order  to  effect  sensitization, 
1  c.c.  injected  by  the  same  route  ten  or  twelve  days  later  would  in  all 
probability  kill  a  half -grown  guinea-pig,  whereas  0.1  c.c.  subcutaneously 
would  be  followed  by  serious  symptoms. 

Rabbit. — Reference  has  been  made  elsewhere  to  the  pioneer  work  of 
Arthus,  who  first  described  the  local  anaphylactic  reaction  about  the 
site  of  subcutaneous  injection.  He  also  described  objectively  the  most 
important  symptoms  of  acute  anaphylactic  death  in  the  rabbit,  as  well 
as  the  more  ordinary  type,  which  ends  in  recovery. 

In  acute  and  fatal  anaphylactic  shock  in  the  rabbit  Auer 1  found  slow 
respiration,  weak  or  absent  heart  action,  with  fall  in  blood-pressure, 
general  prostration,  the  sudden  falling  of  the  animal  on  its  side,  a  short 
clonic  convulsion,  increased  peristalsis,  and  expulsion  in  feces  and  urine. 
Death  is  ascribed  to  a  vascular  or  cardiac  shock  or  to  a  failure  of  the 
heart  action  of  peripheral  origin,  mostly  affecting  the  right  side,  and  due 
to  a  form  of  chemical  vigor.  The  muscle  may  be  gray,  stiff,  very  tough 
to  the  finger-nail,  and  non-irritable.  Further  evidence  of  the  importance 
of  heart  failure  in  anaphylaxis  in  the  rabbit  is  furnished  by  the  electro- 
cardiographic  study  of  Auer  and  Robinson.2  Blood  coagulability  is 
delayed. 

Rabbits  are  by  no  means  so  easily  sensitized  nor  to  so  high  a  degree 
as  guinea-pigs.  Non-fatal  anaphylaxis  accompanied  by  fall  in  blood- 
pressure,  increased  heart-rate,  and  active  intestinal  peristalsis  is  readily 
produced,  but  there  is  considerable  uncertainty  in  inducing  acute 
anaphylactic  death.  Sensitization  is  usually  effected  by  two  or  three 
intravenous  injections  of  1  c.c.  of  serum  at  intervals  of  three  days.  In- 
toxication generally  follows  an  intravenous  injection  of  1  to  5  c.c.  of 
serum  about  four  to  six  weeks  later.  Much  smaller  doses  than  these 
may,  however,  be  used,  as  was  occasionally  shown  during  immuniza- 
tion of  rabbits  with  erythrocytes  for  the  production  of  hemolytic  ambo- 
ceptor,  when  minute  traces  of  serum,  escaping  the  washing  process, 
served  to  sensitize,  and  at  a  later  injection  produced  acute  anaphylactic 
shock  and  death  within  a  very  few  minutes. 

Cats. — Anaphylaxis  in  the  cat  has  been  studied  especially  by 
Schultz,3  who  observed  that  cardiac  disturbances  followed.  Horse 
serum,  however,  was  found  markedly  toxic  in  effect,  even  in  the  un- 

1  Jour.  Exp.  Med.,  1911,  xiv,  476.  2  Jour.  Exp.  Med.,  1913,  xviii,  450. 

3  Jour.  Phar.  and  Exper.  Therap.,  1911-12,  3,  302. 


576  ANAPHYLAXIS 

sensitized  animal,  as  little  as  0.25  c.c.  per  kilo  killing  young,  normal  cats, 
and  0.1  c.c.  per  kilo  causing  a  fall  in  blood-pressure  both  in  the  normal 
and  in  the  sensitized  animals. 

Dogs. — In  these  animals  the  most  constant  symptom  of  anaphy- 
laxis  is  an  initial  and  transitory  rise  in  blood-pressure,  followed  by  a 
prompt  fall  of  from  80  to  100  mm.  of  mercury.  This  was  first  described 
by  Biedl  and  Kraus,1  and  subsequently  by  Eisenbrey  and  Pearce,2 
Robinson  and  Auer.3  The  general  symptoms  are  not  so  violent  as  are 
those  that  occur  in  the  guinea-pig  and  death  is  infrequent.  Following 
intravenous  injection  of  the  intoxicating  dose  of  serum  there  may  be 
great  restlessness,  marked  prostration,  and  vomiting,  tenesmus,  and  in- 
voluntary discharge  of  feces  and  urine.  .  If  death  does  not  occur,  a  con- 
dition of  hemorrhagic  inflammation  in  both  the  large  and  the  small 
intestine  may  develop,  called  by  Richet  "  chronic  anaphylaxis, "  and  by 
Schittenhelm  and  Weichardt,  "enteritis  anaphylactica. "  Robinson 
and  Auer,  by  an  electrocardiographic  study,  detected  cardiac  changes 
consisting  of  disturbance  of  the  heart  impulses,  abnormalities  in  ventric- 
ular contractions,  and  other  interferences  with  the  mechanism  of  the 
heart,  due  probably  to  the  effect  of  horse  serum  on  the  peripheral  car- 
diac tissue,  and  independent  of  the  drop  in  blood-pressure  or  any  effect 
upon  the  central  nervous  system.  The  heart  changes  do  not  appear  to 
exert  a  primary  influence  on  the  blood-pressure,  which  is  due  to  an 
effect  upon  the  splanchnics,  and  is  probably  a  secondary  factor  in  ana- 
phylaxis or  vascular  shock  of  the  dog.  In  many  instances  there  is 
leukopenia,  with  loss  of  mononuclear  cells.  Coagulation  of  the  blood  is 
delayed,  a  condition  first  described  by  Biedl  and  Kraus,4  who  believed 
it  to  be  due,  probably,  to  a  decrease  in  thromboplastin  or  an  excess  of 
antithrombin.  Pepper  and  Krumbharr5  have  shown  that,  by  adding 
small  amounts  of  thromboplastin  to  the  non-coagulating,  post-anaphy- 
lactic,  oxalated  plasma,  the  coagulability  of  the  blood  will  be  restored. 

As  stated  elsewhere,  it  may  be  difficult  to  produce  anaphylaxis  in 
a  dog.  Usually  a  subcutaneous  injection  of  10  c.c.  of  horse  serum, 
followed  in  from  three  to  six  weeks  by  5  c.c.  intravenously,  will  at  least 
cause  a  marked  fall  in  blood-pressure  or  fatal  anaphylaxis. 

Other  Animals. — White  mice  and  rats,  while  they  may  not  develop 
acute  anaphylactic  asphyxia,  such  as  is  observed  in  guinea-pigs,  do  react 

1  Wien.  klin.  Wochenschr.,  1909,  xxii,  365. 

2  Jour.  Pharmacol.  and  Exper.  Therap.,  1912,  iv,  27. 

3  Jour.  Exper.  Med.,  1913,  xviii,  556. 

4  Wien.  klin.  Wochenschr.,  1909,  ii,  363.         5  Jour.  Infect.  Dis.,  1914,  xiv,  476. 


MECHANISM   OF  ANAPHYLAXIS  ,577 

to  horse  serum,  as  was  shown  by  Schultz  and  Jordan.  This  reaction  is 
evidenced  by  restlessness,  marked  irritability  of  the  skin,  involuntary 
passage  of  urine  and  feces,  and  temperature  and  blood-pressure  changes. 
Anaphylactic  reactions  have  also  been  observed  to  occur  in  numerous 
other  animals,  e.  g.,  in  cows,  sheep,  horses,  hens,  pigeons,  and  in 
certain  cold-blooded  animals,  the  symptoms  varying  according  to  the 
species.  These  reactions  have  not  as  yet  been  carefully  studied. 


MECHANISM  OF  ANAPHYLAXIS 

Whereas  the  lesions  and  symptoms  of  anaphylactic  shock  here 
described  in  different  species  of  animals  are  those  commonly  observed 
with  serum  proteins,  they  vary  in  no  essential  when  any  protein  agent 
is  used  when  the  conditions  of  dosage  and  administration  are  the  same. 
It  is  evident,  however,  that  no  one  symptom,  or  group  of  symptoms, 
can  be  regarded  as  characteristic  of  anaphylaxis  in  all  animals.  The 
various  species  present  widely  differing  pictures  with  the  same  protein 
substance,  and  these  differences  are  best  explained  on  the  ground  of 
changes  in  the  anatomic  structure  and  physiologic  reaction  of  different 
animals.  Thus,  Schultz  has  shown  that  serum  anaphylaxis  is  essentially 
a  matter  of  hypersensitization  of  smooth  muscle  in  general,  and  that, 
during  anaphylactic  shock,  all  smooth  muscle  contracts.  In  the  guinea- 
pig  this  effect  is  most  evident  in  the  bronchi,  owing  to  the  peculiar, 
though  normal,  anatomic  structure  of  the  mucosa,  which  is  relatively 
thick  as  compared  with  the  lumen,  so  that  contraction  of  the  smooth 
muscle  throws  it  into  folds  that  completely  occlude  the  bronchi  causing 
death  from  inspiratory  asphyxia.  The  bronchial  mucosa  of  dogs, 
rabbits,  and  rats,  however,  is  relatively  thin  and  poor  in  smooth  muscle 
tissue,  which  may  account  for  an  entire  absence  of  transitory  respiratory 
difficulties  during  anaphylactic  shock  in  these  animals.  In  the  dog  the 
most  marked  effect  is  apparent  upon  the  smooth  muscle  of  the  gastro- 
intestinal tract,  contraction  resulting  in  setting  up  vigorous  intestinal 
peristalsis,  vomiting,  and  involuntary  emptying  of  the  urinary  bladder. 
The  characteristic  initial  rise  in  blood-pressure  may  be  due  to  con- 
striction of  the  splanchnic,  pulmonary,  coronary,  and  systemic  arteries, 
followed  by  a  condition  of  paresis  and  a  fall  in  blood-pressure.  The 
cardiac  muscle  is  also  involved,  particularly  on  the  right  side,  as  shown 
by  Robinson  and  Auer,  and  this  favors  a  venous  accumulation  of  blood. 
In  the  rabbit  a  similar  effect  is  noted  upon  the  smooth  muscle  of  the 
blood-vessels,  and  particularly  on  the  heart,  as  well  as  upon  the  gastro- 
37 


578  ANAPHYLAXIS 

intestinal  tract.  Our  present  knowledge  would  ascribe  these  effects, 
therefore,  to  a  local  or  peripheral  action  of  the  protein  upon  smooth 
muscle,  and  not  primarily  on  the  central  nervous  tissues,  as  was  originally 
believed. 

The  fall  in  blood-pressure,  therefore,  appears  to  be  a  most  constant 
and  primary  factor.  So  far  as  I  am  aware,  no  blood-pressure  studies 
on  the  anaphylactic  guinea-pig  have  been  made.  In  this  animal  the 
heart  continues  to  beat  after  respiration  ceases,  but  this  phenomenon 
may  be  due  to  mechanical  and  other  factors  dependent  upon  the  extreme 
pulmonary  emphysema.  Fall  in  blood-pressure  and  congestion  of  the 
splanchnic  area  may  produce  cerebral  anemia,  and  be  responsible  in  some 
measure  for  the  respiratory  disturbances,  the  retching,  the  involuntary 
expulsion  of  urine  and  feces,  the  great  depression  and  muscular  weakness, 
and  the  speedy  recovery  when  death  does  not  result. 

In  man  the  marked  urticarial  and  other  rashes  and  the  inspiratory 
asthma  of  those  peculiarly  sensitive  to  a  protein  due  to  a  narrowing  of 
the  bronchi,  the  latter  being  analogous  to  the  condition  observed  in 
the  guinea-pig,  the  diarrhea,  and  the  secondary  drop  in  blood-pres- 
sure, all  indicate  a  similar  action  on  smooth  muscle.  This  also  pro- 
vides an  adequate  pharmacologic  explanation  of  the  action  of  atropin, 
sedatives,  and  anesthetics  in  alleviating  or  masking  the  symptoms  of 
acute  anaphylaxis.  (See  Chapter  XXVIII.) 

Aside  from  the  severe  fall  in  blood-pressure  and  temperature,  other 
effects  of  anaphylaxis  are  leukopenia,  local  and  general  eosinophilia 
(Vaughan,1  Moschowitz,2  Schlecht  and  Schwenket3),  and  reduced 
coagulability  of  the  blood.  Pfeiffer4  found  'poisonous  substances  in 
the  urine  during  anaphylactic  intoxication,  and  Hirschfeld5  detected  a 
pressor  substance  in  the  serum  of  intoxicated  guinea-pigs. 

A  more  critical  study  of  the  nature  and  varieties  of  anaphylactogens, 
or  substances  capable  of  producing  anaphylactic  sensitization,  will  now 
be  made.  This  will  include  also  a  consideration  of  the  nature  of  the 
substances  directly  responsible  for  the  anaphylactic  intoxication,  com- 
monly known  as  anaphylactotoxins,  and  of  the  question  as  to  whether 
anaphylactic  intoxication  is  the  result  of  an  interaction  in  the  blood- 
stream (humoral)  or  in  the  cells  (histogenetic)  or  in  both. 

1  Zeitschr.  f.  Immunitatsf.,  1911,  9,  458. 

2  New  York  Med.  Jour.,  January  7,  1911. 

a  Arch.  exp.  Path.  u.  Pharm.,  1912,  68,  163. 
4  Zeitschr.  f.  Immunitatsf.,  1911,  10,  550. 
s  Zeitschr.  f.  Immunitatsf.,  1912,  14,  466. 


ANAPHYLACTOGENS,  OR  ALLERGENS  579 

ANAPHYLACTOGENS,  OR  ALLERGENS 

So  far  as  is  now  known,  only  proteins  may  become  anaphylactogens, 
and  with  the  exception  of  gelatin  and  a  few  other  proteins,  practically 
any  soluble  protein  will  produce  sensitization  and  intoxication  of  sus- 
ceptible animals.  Bacterial  substances,  extracts  of  plant  tissues, 
purified  vegetable  proteins,  and  proteins  derived  from  invertebrates  and 
cold-blooded  vertebrates  have  all  been  found  capable  of  acting  as 
anaphylactogens  when  introduced  in  a  soluble  and  unaltered  condition 
into  an  animal. 

The  proteins  concerned  must  be  foreign  to  the  circulating  blood  of 
the  injected  animal,  but  they  may  be  tissue  proteins  of  the  same  animal — 
e.  g.,  syncytial  cells — that  are  not  normally  present  in  the  blood.  Indeed, 
Uhlenhuth  and  Haendel1  claimed  to  have  sensitized  a  guinea-pig  with 
the  dissolved  lens  of  one  eye  so  that  it  reacted  to  a  subsequent  injection 
of  the  lens  of  the  other  eye.  Proteins  in  solution  are  more  active  than 
those  in  suspension  or  in  partial  solution,  and  in  general  tissue  proteins 
are  less  active  than  proteins  in  the  blood,  lymph,  and  secretions,  but 
even  keratins  may  produce  anaphylaxis  when  dissolved  (Krusins2), 
Uhlenhuth3  has  obtained  positive  results  with  proteins  from  mummies. 
As  previously  stated,  the  altered  protein  of  an  animal  may  be  reinjected 
again  into  the  animal  and  induce  an  anaphylactic  reaction.  Recently 
Richet4  has  directed  attention  to  this  phenomenon,  which  he  calls 
"indirect  anaphylaxis,"  through  observing  an  intense  leukocytosis  in  a 
dog  which  reached  the  maximum  on  the  eighth  day  following  a  second 
chloroformization. 

Non-protein  Anaphylactogens. — As  with  other  immunologic  reac- 
tions, observations  have  been  made  that  are  interpreted  as  indicating 
that  non-protein  substances  are  capable  of  producing  anaphylaxis; 
thus  Pick  and  Yamanouchi5  sought  to  demonstrate  the  antigenic 
properties  of  alcohol-soluble  constituents  of  horse  and  beef  serum,  but 
conservatively  concluded  that  their  results  may  have  been  due  to  a  com- 
bined action  of  protein  and  fat  combinations.  Similar  conclusions  were 
also  drawn  by  Uhlenhuth  and  Haendel6  in  their  study  of  animal  and 


1  Zeitschr.  f.  Immunitatsf.,  1910,  4,  761. 

2  Arch.  f.  Augenheilk.,  Suppl.,  1910,  47,  47. 

3  Zeitschr.  f.  Immunitatsf.,  1910,  4,  774. 

4  Quoted  in  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  711. 

5  Zeitschr.  f .  Immunitatsf.,  1909,  5,  676. 

6  Zeitschr.  f.  Immunitatsf.,  1910,  iv,  761. 


580  ANAPHYLAXIS 

vegetable  oils  and  fats.  Bogomolex1  is  less  conservative,  and  believes 
that  he  has  succeeded  in  producing  lipoid  anaphylaxis;  these  claims, 
however,  could  not  be  confirmed  by  Thiele  and  Embleton.2  Meyer3 
was  able  to  sensitize  pigs  with  pure  lipoids  extracted  from  tape-worms, 
but  was  unable  to  intoxicate  them  with  the  same  extracts,  results  that 
may  be  understood,  since  White  and  Avery4  have  shown  that  as  little 
as  0.0001  milligram  of  edestin  will  serve  to  sensitize  a  pig,  whereas 
larger  amounts  of  protein — more  than  is  contained  in  Meyer's  prepara- 
tions— are  necessary  to  produce  intoxication.  Finally,  the  studies  of 
Wilson5  and  White6  leave  no  doubt  as  to  the  fact  that  pure  lipoids 
cannot  produce  anaphylaxis. 

It  is  possible,  however,  for  non-protein  substances  to  combine  with 
or  alter  the  proteins  of  an  animal  and  thus  cause  anaphylaxis.  In  this 
way  can  be  explained  apparent  anaphylactic  reactions  to  iron,  salvarsan, 
iodin,  arsenic  compounds,  and  other  medicinal  agents. 

Chemistry  of  Protein  Anaphylactogens. — The  purest  known  proteins 
act  as  anaphylactogens  or  sensitizers;  in  fact,  the  purer  the  protein, 
the  more  thoroughly  it  sensitizes  the  animal  and  the  smaller  is  the  dose 
necessary  to  produce  intoxication.  The  crystallized  proteins  of  hemo- 
globin, egg-albumen,  and  such  pure  vegetable  proteins  as  edestin  and 
excelsin,  are  powerful  sensitizers.  According  to  Wells,7  nothing  less 
than  an  entire  protein  molecule  will  suffice  to  produce  anaphylaxis, 
although  Zunz 8  claims  to  have  observed  typical  reactions  with  the  pro- 
teoses  of  fibrin,  and  Abderhalden9  obtained  one  with  a  synthetic  poly- 
peptid.  It  is  not  necessary,  however,  for  a  protein,  in  order  to  be  active, 
to  contain  all  the  known  amino-acids  of  proteins,  for  certain  vegetable 
proteins,  e.  g.,  hordein  and  gliadin,  which  lack  one  or  more  amino-acids, 
such  as  glycocoll  or  tryptophane,  may  produce  typical  reactions.  Pre- 
sumably, the  inability  of  pseudoproteins,  such  as  gelatin,  to  act  as 
anaphylactogens  depends  upon  their  deficiency  in  aromatic  radicals. 

Wells  has  obtained  negative  results  with  purified  nucleoproteins,  as 
well  as  with  the  isolated  components  of  nucleins,  such  as  histon  and 
nucleic  acid. 

1  Zeitschr.  f.  Immunitatsf.,  1910,  v,  121,  ibid,  1910,  vi,  332. 

2  Zeitschr.  f.  Immunitatsf.,  1913,  xvi,  160. 

3  Folia  Serologica,  1911,  vii,  771;  Zeitschr.  f.  Immunitatsf.,  1914,  xxi,  654. 

4  Jour.  Infect.  Dis.,  1913,  xiii,  103. 

6  Jour.  Path,  and  Bact.,  1913,  xviii,  163. 

6  Jour.  Med.  Res.,  1914,  xxx,  383.  7  Jour.  Infect.,  Dis.  1913,  xii,  341. 

8  Zeitschr.  f.  Immunitatsf.,  1913,  60,  580. 

9  Zeitschr.  physiol.  Chem.,  1912,  81,  314. 


ANAPHYLACTOGENS,  OR  ALLERGENS  581 

While,  therefore,  it  is  probable,  although  it  has  not  been  definitely 
proved,  that  nothing  less  than  the  entire  protein  molecule  is  capable  of 
producing  the  typical  reaction,  the  questions  arise  whether  the  whole 
molecule,  or  only  a  certain  group  thereof,  determines  the  specificity, 
and  whether  the  whole  molecule,  or  only  a  portion,  is  concerned  as  the 
sensitizing  agent.  It  is  now  generally  accepted  that  both  the  sensitizing 
and  the  intoxicating  agents  are  one  and  the  same  protein,  and  the  older 
view,  which  held  that  in  a  mixed  protein  substance,  such  as  blood-serum, 
corpuscles,  egg-albumen,  etc.,  one  protein  is  present  that  sensitizes  and 
another  that  intoxicates,  is  probably  erroneous.  Besredka,  for  instance, 
finds  that  when  a  protein  used  to  produce  intoxication  is  heated  it  is  less 
likely  to  prove  fatal,  and  he  concludes  that  proteins  contain  a  thermosta- 
bile  sensitizing  and  a  thermolabile  intoxicating  portion.  Doerr  and 
Russ,  however,  have  shown  by  carefully  conducted  experiments  that 
heat  affects  both  properties  of  proteins  to  the  same  degree.  Since  pure 
proteins,  as,  e.  g.,  highly  purified  edestin,  which  is  believed  to  be  a  chem- 
ical unit,  act  as  exquisite  sensitizers  and  intoxicants,  it  seems  reasonable 
to  believe  that  the  sensitizing  and  poisonous  group  are  constituents  of 
the  same  protein  substance.  Whether  or  not  both  sensitizing  and 
intoxicating  groups  are  contained  in  each  single  molecule  of  a  pure  pro- 
tein is  a  question  that  cannot  be  answered  until  we  can  be  certain  that 
absolutely  pure  proteins  are  secured  to  start  with,  and  until  our  methods 
of  effecting  its  cleavage  have  been  perfected.  Vaughan  and  his  cowork- 
ers  have  long  maintained  that  a  sensitizing  non-poisonous  and  a  non- 
sensitizing  toxic  portion  are  groups  of  the  same  molecule,  which  they  are 
able  to  obtain  in  vitro  from  animal,  bacterial,  and  vegetable  proteins  by 
a  method  of  splitting  with  sodium  hydroxid  in  absolute  alcohol,  as 
described  in  the  chapter  on  Infection.  The  toxic  intramolecular  group 
is  regarded  as  non-specific,  and  the  same  for  all  proteins,  which  explains 
the  identity  of  the  symptoms  of  anaphylactic  shock  whatever  the  pro- 
tein by  which  it  is  induced.  The  non-toxic  sensitizing  group,  however, 
is  specific,  although  it  may  not  itself  be  a  protein,  or  at  least  a  biuret 
body.  Whether  or  not  all  proteins  contain  a  sensitizing  group  has  not 
been  determined.  In  keeping  with  his  theory  of  the  role  of  the  toxic 
moiety  of  a  split  protein  molecule  in  the  production  of  disease,  Vaughan 
believes  that  when  proteins  are  introduced  parenterally  into  animals, 
the  non-toxic  portion  stimulates  the  body-cells  to  elaborate  specific  fer- 
ments, constituting  the  phase  of  sensitization,  so  that  when  this  protein 
is  subsequently  introduced,  digestion  rapidly  takes  place  with  the 
liberation  of  the  toxic  substance  responsible  for  the  characteristic  symp- 


582  ANAPHYLAXIS 

toms,  which  may  terminate  in  death.  This  interesting  and  plausible 
theory  will  be  referred  to  again.  It  has  received  further  experimental 
support  from  the  work  of  White  and  Avery1  with  split  edestin  and 
split  tubercle-cell  substance;2  and  from  that  of  Zunz3  and  Wells  and 
Osborne,4  the  last-named  observers  working  with  vegetable  proteins, 
and  concluding  that  although  it  is  probable  that  the  entire  protein  molecule 
is  involved  in  the  anaphylactic  reaction,  only  certain  groups  are  specifically 
concerned  in  the  process.  In  other  words,  it  would  appear  that  anaphy- 
laxis, — for  example,  serum  anaphylaxis — is  not  due  to  one  protein  sub- 
stance in  the  serum  that  sensitizes  and  another  that  intoxicates,  both 
properties  residing  in  the  same  protein  molecule.  Whether  they  are  the 
same  intramolecular  substances  existing  side  by  side, — one  the  sensitizer 
and  the  other  the  intoxicator, — as  is  believed  by  Vaughan,  cannot  be 
definitely  decided,  although  experimental  work  would  tend  to  indicate 
that  the  latter  may  be  the  true  explanation. 

Physical  State  of  Anaphylactogens. — The  results  of  experiments 
all  tend  to  support  the  theory  that  proteins  in  solution  are  most  power- 
ful in  producing  anaphylaxis,  because  they  are  able  to  come  into  intimate 
contact  with,  body-cells,  and  cell  permeation  is  probably  necessary  for 
the  most  complete  sensitization.  This  explains  in  part  the  conflicting 
statements  concerning  the  effect  of  heat  on  the  sensitizing  properties  of 
blood-serum.  Rosenau  and  Anderson  found  that  animals  could  not  be 
sensitized  with  serum  that  has  been  heated  at  100°  C.,  whereas  Doerr 
and  Russ  placed  the  point  at  80°  C.  Besredka  showed  that  the  sensitizing 
properties  are  in  part  at  least,  dependent  upon  the  physical  condition  of 
the  protein,  and  that  heating  undiluted  blood-serum  coagulates  the  pro- 
tein and  leads  to  a  decrease  of  its  anaphylactogenic  properties.  Similarly, 
Vaughan  found  that  proteins  that  were  insoluble  in  water,  as,  for  ex- 
ample, edestin,  sensitize  more  readily  when  dissolved  in  salt  solution. 
The  same  factors  are  operative  with  the  protein  used  for  intoxication, 
the  physical  state  of  the  protein  substance  having  a  direct  bearing 
on  the  rapidity  with  which  shock  is  produced.  Temperatures  high 
enough  to  disrupt  and  destroy  proteins  are,  however,  equally  destructive 
to  their  sensitizing  properties. 

An  interesting  question  in  this  connection  is  whether  sensitization 
and  intoxication  may  occur  with  the  parenteral  introduction  of  protein 
as  with  the  food.  In  the  great  majority  of  instances  the  gastro-in- 
testinal  enzymes  so  completely  disrupt  the  protein  molecule  that  sen- 

1  Jour.  Inf.  Dis.,  1913,  xiii,  103.  2  Jour.  Med.  Res.,  1912,  xxvi,  317. 

3  Zeitschr.  f.  Immunitatsf.,  1913,  xvi,  580.       4  Jour.  Inf.  Dis.,  1913,  xii,  341. 


ANAPHYLACTOGENS,  OR  ALLERGENS  583 

sitization  and  intoxication  do  not  occur.  Guinea-pigs  have,  however, 
been  sensitized  by  feeding  them  meat  or  serum,  and  instances  of  buck- 
wheat, fish,  and  egg  idiosyncrasies  would  tend  to  indicate  that  intoxica- 
tion may  result  from  the  ingestion  of  these  substances  in  sensitive 
persons. 

Rosenau  and  Amos1  have  demonstrated  that  proteins  in  a  volatile 
state,  as  in  the  exhaled  breath  of  men,  when  condensed  and  injected  into 
guinea-pigs  will  sensitize  these  animals  to  subsequent  injections  of  human 
serum.  While  it  is  doubtful  if  the  complex  molecule  possesses  the  power 
of  passing  into  the  air  in  a  gaseous  form,  it  may  probably  exist  in  col- 
loidal solution.  Rosenau  was  also  able,  by  keeping  guinea-pigs  in 
stables  together  with  horses,  to  sensitize  them  to  horse  serum.  These 
experiments  are  of  fundamental  importance  in  explaining  instances  of 
human  anaphylactic  phenomena  among  those  sensitive  to  horse  protein, 
— as,  e.  g.,  persons  seized  with  sneezing  and  asthma  when  they  come 
near  horses, — and  also  tend  to  show  how  minute  may  be  the  quantity 
of  protein  capable  of  sensitizing  and  intoxicating  body-cells. 

Bacterial  Anaphylactogens. — All  bacterial  proteins  .are  anaphy- 
lactogens,  although,  on  account  of  the  physical  state  of  the  bacteria, 
they  yield  reactions  more  irregular  and  weaker  than  those  observed 
with  proteins  in  solution.  The  tuberculin,  luetin,  mallein,  and  similar 
reactions  are  true  anaphylactic  phenomena.  Rosenau  and  Anderson, 
Vaughan  and  Wheeler,  Kraus,  and  others  have  observed  anaphylactic 
reactions  with  various  bacteria,  such  as  Bacillus  subtilis  and  colon, 
typhoid,  anthrax,  and  tubercle  bacilli.  Not  infrequently  reactions 
occur  during  the  therapeutic  administration  of  tuberculin  and  bacterial 
vaccines. 

This  brings  up  the  interesting  question  as  to  whether  toxins  are 
anaphylactogens,  a  subject  previously  mentioned  in  the  historic  review 
of  this  subject.  Instances  of  hypersensitiveness  to  diphtheria  and  te- 
tanus toxins  were  early  observed  in  attempts  to  immunize  horses  in  the 
production  of  antitoxins.  As  it  is  extremely  difficult,  if  not  impossible, 
to  isolate  a  toxin  free  from  other  constituents  of  the  medium  into  which 
it  was  excreted  by  microorganisms,  this  question  cannot  be  answered 
in  a  definite  manner.  There  is  no  direct  proof,  however,  that  toxins 
sensitize,  although  the  protein  in  the  toxin  filtrate  may  serve  to  do  so. 
In  1902  Vaughan  and  Gelston2  showed  that  the  poison  contained  in  the 
cellular  substance  of  the  diphtheria  bacillus  is  an  entirely  different  one 

1  Jour.  Med.  Research,  1911,  xxv,  35. 

2  Trans.  Assoc.  Amer.  Phys.,  1902,  17,  308. 


584  ANAPHYLAXIS 

from  the  toxin  elaborated  by  the  same  microorganism,  results  that  were 
confirmed  in  1911  by  Friedberger  and  Reiter,1  working  with  the  dysen- 
tery bacillus.  Thus  hypersensitiveness  to  toxins  is  probably  not  an 
anaphylactic  phenomenon,  but  is  due  to  a  greater  affinity  of  the  body- 
cells  for  the  toxin.  This  explains  the  so-called  paradox  of  Kretz,  who 
found  that  while  the  injection  of  an  accurately  neutralized  toxin-anti- 
toxin mixture  produces  no  bad  results  in  a  normal  animal,  in  one  that 
has  been  previously  actively  immunized  with  toxin,  the  reverse  occurs. 
Apparently  the  sessile  receptors  have  a  stronger  affinity  for  toxin  than 
have  the  free  receptors,  and  accordingly  the  toxin  becomes  dissociated 
and  combines  with  the  cells. 

This  view  is  also  substantiated  by  the  observation  that  the  symptoms 
of  intoxication  caused  by  the  toxin  used  for  immunization  are  not  those 
of  anaphylaxis,  which,  for  a  certain  animal,  are  the  same  regardless  of 
the  source  of  the  protein.  Toxin  hypersensitiveness  does  not  seem  to 
be  transmissible  to  normal  animals,  whereas  in  anaphylaxis  the  condi- 
tion may  be  transmitted  (passive  anaphylaxis). 

Whether  or  not  endotoxins  act  as  anaphylactogens  cannot  be  definitely 
stated.  If  they  do,  their  action  and  effects  are  intimately  connected 
with  those  ascribed  to  the  protein  contained  in  the  bacterial  cell.  It  is 
unlikely,  however,  that  they  play  any  role  in  inducing  hypersuscepti- 
bility,  as  their  toxicity  is  usually  apparent  soon  after  injection,  and 
before  sensitization  has  occurred. 

The  necessary  period  of  incubation  between  sensitization  and  in- 
toxication, the  symptoms,  and  the  fact  that  in  some  cases  an  immunity 
is  induced,  are  results  that  strengthen  the  belief  that  the  phenomenon  of 
hypersensitiveness  has  a  practical  significance  in  the  prevention  and  cure 
of  certain  infectious  diseases. 


THE  HUMORAL  OR  ANAPHYLATOXIN  THEORIES  OF  ANAPHYLAXIS 

The  symptoms  of  anaphylaxis  that  follow  injection  of  the  protein 
into  an  animal  previously  sensitized  with  the  same  protein  have  been 
ascribed  to  the  effects  of  a  poison  as  the  etiologic  factor,  although  as  yet 
this  poison  has  not  been  isolated  in  a  pure  state. 

The  Anaphylatoxin  or  Protein  Poison. — Vaughan  and  Wheeler 
were  the  first  to  demonstrate  a  poison  in  vitro  by  splitting  proteins  with 
a  solution  of  NaOH  in  absolute  alcohol.  Friedmann  was  first  to  pro- 
duce it  in  vitro  through  the  action  of  ferments  contained  in  immune 
and  normal  rabbit  serum  upon  ox  corpuscles  and  serum  precipitates. 
1  Zeitschr.  f.  Immunitatsf.,  1911,  11,  493. 


ANAPHYLATOXIN    (PROTEIN    POISON)  585 

Weichardt  also  produced  it  by  digesting  placental  protein  with  the 
serum  of  rabbits  immunized  with  placental  cells.  Friedberger1  studied 
the  subject  more  extensively  by  observing  the  action  of  normal  guinea- 
pig  serum  upon  serum  precipitates,  and  was  first  to  apply  the  term 
"anaphylatoxin"  to  the  poison.  At  the  time  he  regarded  it  as  a  true 
toxin,  similar  to  diphtheria  and  tetanus  toxins,  but  at  present  we  know 
that  this  poison  is  not  a  true  toxin,  because  it  cannot  produce  an  anti- 
toxin, is  thermostabile  in  acid  solution,  and  is  not  a  single  specific 
substance,  but  a  mixture  of  more  or  less  closely  related  substances 
in  the  nature  of  protein  cleavage  products,  as  first  shown  by  Vaughan 
and  Wheeler,  and  since  accepted  by  Friedberger  himself  and  a  number 
of  other  investigators.  In  a  strict  sense,  therefore,  this  term  is  a  mis- 
nomer, but  it  is  in  such  general  use  that  it  need  not  be  discarded  if 
we  have  a  clear  understanding  that  the  sum  total  of  independent  re- 
searches by  numerous  investigators  shows  that  it  is  not  a  true  toxin, 
as  tetanus  toxin,  for  instance,  but  a  protein  poison. 

As  the  symptoms  of  anaphylaxis  are  always  the  same  in  the  same 
animal,  no  matter  what  protein  is  used,  it  would  appear  that  the  protein 
poison  is  either  always  the  same  or  composed  of  a  group  of  very  closely 
allied  products,  and,  indeed,  this  seems  to  have  been  proved  by  an 
extended  series  of  researches  with  the  most  diverse  proteins  of  animal, 
bacterial,  and  vegetable  origin. 

It  may  be  stated,  therefore,  that  anaphylatoxins  may  be  regarded  as 
protein  poisons  composed  of  protein  cleavage  products,  and  that  these  are 
responsible  for  the  lesions  and  symptoms  of  anaphylaxis.  There  is  some 
difference  of  opinion  regarding  the  source  of  the  protein  matrix  and  the 
mechanism  of  its  cleavage  with  the  production  of  the  poison  in  anaphy- 
laxis, and  I  shall  consider  this  phase  of  the  subject  later.  There  is, 
however,  a  striking  uniformity  of  experimental  evidence  and  opinion 
regarding  the  role  and  primary  importance  of  the  protein  cleavage 
poisons  in  the  anaphylactic  process.  Briefly  summarized,  the  evidence 
on  this  point  is  as  follows : 

1.  As  was  just  stated,  the  first  to  advance  the  theory  regarding  the 
role  of  protein-split  products  in  anaphylaxis,  as  well  as  in  infection  and 
immunity  in  general,  were  Vaughan  and  Wheeler.  In  1907  these 
observers  showed  that  proteins  may  be  split  by  boiling  with  alcoholic 
sodium  hydroxid  solution  into  two  fractions — one  non-toxic  and  alcohol 
soluble  and  the  other  toxic  and  alcohol  insoluble.  The  toxic  fraction, 
when  injected  into  normal  guinea-pigs  in  doses  of  from  8  to  100  mg., 
1  Zeitschr.  f.  Immunitatsf.,  1910,  4,  636. 


586  ANAPHYLAXIS 

kills  the  animals,  death  being  preceded  by  all  the  symptoms  of  acute 
anaphylactic  intoxication.  This  toxic  moiety  has  been  obtained  by 
this  method  from  the  most  diverse  proteins,  and  seems  to  be  the  same  for 
all,  the  specificity  residing  in  the  non-toxic  attached  group.  This  and 
other  observations  led  Vaughan  and  his  collaborators  to  formulate  the 
hypothesis  that  the  non-toxic  and  specific  intramolecular  group  of  a 
protein  serves  to  stimulate  the  body-cells  to  produce  specific  proteolytic 
enzymes,  and  that  upon  the  injection  of  a  second  dose  of  the  same 
protein,  these  enzymes  at  once  disintegrate  it,  setting  free  the  toxic 
group  that  produces  the  lesions  and  symptoms  of  acute  anaphylactic 
intoxication.  This  protein  cleavage  in  vivo  is  entirely  analogous  to 
the  cleavage  process  occurring  in  vitro  with  the  alcoholic  sodium  hydroxid 
solution,  and  the  poison  in  both  instances  is  regarded  as  the  same. 

2.  Mention  has  previously  been  made  of  the  protein  poison  obtained 
by'  Friedmann  by  digesting  ox  corpuscles  with  immune  and  normal 
rabbit  serum,  and  by  Weichardt  as  the  result  of  the  digestion  of  placental 
protein  with  immune  rabbit  serum.  In  1910  Friedberger,1  by  digesting 
a  serum  precipitate  with  normal  guinea-pig  serum,  obtained  a  similar 
toxic  substance;  by  means  of  ferments  he  secured  a  protein  cleavage 
poison  that,  when  injected  into  normal  guinea-pigs,  produced  the 
symptoms  of  acute  anaphylaxis,  the  process  being  entirely  analogous  to 
that  obtained  by  Vaughan  and  Wheeler  by  protein  splitting  with  alcoholic 
sodium  hydroxid  solution.  Subsequent  studies  by  Friedberger  and  his 
collaborators 2  showed  that  similar  protein  poisons  could  be  obtained  by 
digesting  microorganisms,  as,  e.  g.,  Bacillus  prodigiosus,  Bacillus  typho- 
sus,  and  Bacillus  tuberculosis,  with  normal  guinea-pig  serum.  Fried- 
berger and  Nathan 3  obtained  the  poison  by  digesting  normal  horse  serum 
with  fresh  guinea-pig  serum;  Bordet,4  by  digesting  agar  with  fresh 
serum;  Dold  and  Aoki,5  by  digesting  meningococci,  streptococci, 
pneumococci,  gonococci,  and  various  other  microorganisms  with  fresh 
normal  gunea-pig  serum.  As  was  expected,  cleavage  could  be  facili- 
tated and  hastened  by  using  immune  serum  specific  for  any  particular 
bacterial  or  other  protein. 

These  results  indicate  that  fresh  normal  guinea-pig  serum  contains  a 
normal  thermolabile  non-specific  proteolytic  ferment  capable  of  split- 
ting some,  through  probably  not  all,  proteins,  a  view  advanced  by 

1  Zeitschr.  f.  Immunitatsf.,  1910,  4,  636. 

2  Zeitschr.  f.  Immunitatsf,,  1911,  9,  369. 
.    3  Zeitschr.  f.  Immunitatsf.,  1911,  9,  567. 

4  Compt.  rend  de  Soc.  de  Biol.,  1913,  Ixxiv,  No.  5. 
6  Zeitschr.  f.  Immunitatsf.,  1912,  12,  200. 


ANAPHYLATOXIN  (PROTEIN    POISON)  587 

Vaughan  many  years  previously.  After  sensitization,  a  specific  proteo- 
lytic  ferment  is  produced  for  the  particular  protein  injected,  the  presence 
of  the  specific  in  addition  to  the  normal  ferments  constituting  the  differ- 
ence between  normal  and  sensitized  animals. 

Since  these  studies  show  that,  when  incubated  with  normal  serum, 
bacteria  and  certain  other  proteins  yield  a  soluble  and  active  poison, 
the  question  naturally  arises  why  this  reaction  does  not  occur  when 
these  proteins  are  first  injected  directly  into  the  blood?  Vaughan  has 
answered  this  question  by  assuming  that  the  ferment  is  in  a  more  avail- 
able form  in  the  serum  than  it  is  in  the  blood,  since  the  ferment  is  prob- 
ably largely  a  leukoprotease  mainly  derived  from  the  disintegration  of 
leukocytes.  Or  it  may  be  that  the  cleavage  is  carried  on  in  the  circulat- 
ing blood  beyond  the  point  of  the  products  constituting  the  protein 
poison,  or  that  the  inclusion  of  the  foreign  protein  by  the  phagocytes  may 
delay  the  disruption  of  the  former. 

The  Protein  Matrix. — While  there  is  this  consensus  of  opinion 
among  the  adherents  of  the  chemical  or  anaphylatoxin  theory  of  ana- 
phylaxis  regarding  the  role  of  protein  poison  in  the  production  of  ana- 
phylaxis,  there  is  some  diversity  of  opinion  regarding  the  source  of  the 
protein  matrix,  i.  e.,  whether  the  protein  that  is  broken  down  is  the 
protein  injected  or  the  protein  of  the  person's  own  serum,  especially 
in  view  of  the  fact  that  mixtures  of  kaolin  and  normal  guinea-pig  serum 
produce  the  poison  in  vitro.  It  has  been  suggested — (a)  That  the 
kaolin,  agar,  bacteria,  etc.,  absorb  the  complement  from  the  serum, 
and  that  this  renders  the  serum  poisonous;  (6)  that  the  poison  is 
preformed  in  the  serum,  but  that  its  action  is  neutralized  by  some 
other  constituent  of  the  serum  that  is  absorbed;  (c)  that  the  absorption 
of  some  constituent  of  the  serum  leads  to  a,  breaking-up  of  serum  pro- 
teins, with  liberation  of  the  poison.  The  latter  view,  as  shown  by  Job- 
ling  and  Petersen, 1  appears  to  be  the  most  plausible.  These  writers 
believe  that  the  normal  tryptic  or  proteolytic  ferment  of  the  blood  is 
held  in  check  by  an  antiferment  of  the  nature  of  unsaturated  fatty  acids, 
and  that  laokin,  bacteria,  agar,  etc.,  remove  this  antitryptic  influence 
by  absorbing  the  lipoidal  antiferment,  setting  the  ferment  free,  which 
then  acts  upon  the  serum  protein,  producing  the  toxic  protein  poison 
(sero toxin).  While  Vaughan,  Friedberger,  and  their  collaborators 
believe  that  the  protein  poison  is  derived  from  the  injected  protein, 
Jobling  and  Petersen  believe  that  the  matrix  is,  indeed,  the  protein  of 
the  animal's  own  serum.  Doerr2  likewise  regards  it  as  highly  improbable 

1  Jour.  Exper.  Med.,  1914,  xix,  459  and  480. 

2  Handb.  d.  path.  Mikroorgan.,  Kolle  and  Wassermapn,  second  edition-,  ii,  947. 


588  ANAPHYLAXIS 

that  the  poison  is  generated  by  the  bacteria  or  other  foreign  protein, 
but  believes  that  it  is  derived  from  the  serum  through  the  absorption  of 
inhibitory  antibodies  by  the  bacteria,  precipitates,  etc.  There  can  be 
no  doubt,  however,  of  the  high  specificity  of  the  anaphylactic  reaction, 
which  indicates  that  specific  ferments  are  produced  for  the  protein 
injected,  and  while  the  discussion  cannot  be  considered  closed,  it  would 
appear  that,  for  the  present,  it  would  be  best  to  accept  the  theory  that 
anaphylaxis  is  due  both  to  the  cleavage  of  the  injected  anaphylactogen 
and  the  blood-serum  by  normal  and  specific  proteolytic  ferments. 

The  Anaphylactin  (Allergin)  or  Anaphylactic  Antibody. — There  is  a 
diversity  of  opinion  concerning  the  nature  of  the  substance  developed 
in  the  body  under  the  influence  of  the  anaphylactogen,  which  is  capable 
of  cleaving  the  latter  and  producing  the  anaphylactic  poison.  That 
such  a  body  exists  in  the  blood  of  the  sensitized  animal  is  shown  by  the 
production  of  passive  anaphylaxis  in  normal  animals  by  injecting  into 
them  a  few  cubic  centimeters  of  blood  or  serum  from  a  sensitized  animal. 
The  animal  so  sensitized  becomes  at  once  or  within  a  few  hours  sensitive 
to  the  specific  anaphylactogen,  regardless  of  the  species  of  animal 
furnishing  the  immune  serum. 

Terminology. — Various  names  have  been  applied  to  this  body. 
Vaughan  objects  to  the  term  antibody,  although  this  designation  would 
seem  to  be  appropriate  if  we  consider  that  it  is  a  reactionary 
product  or  antibody  to  the  protein  responsible  for  its  production,  al- 
though, instead  of  being  protective  to  the  animal,  its  host,  it  is  just  the 
reverse.  Richet  named  it  toxogen,  because  it  is  responsible  for  the  pro- 
duction of  a  poison.  Otto  speaks  of  it  as  reaction  body,  Nicolle  as  an 
albuminolysin,  Besredka  as  sensibiUsin,  and  von  Pirquet  as  oiler  gin. 

Nature  of  Anaphylactin. — A  full  discussion  of  the  nature  of  this  body, 
which  is  believed  to  cleave  the  anaphylactogen  and  set  free  the  anaphy- 
latoxin  or  protein  poison,  would  involve  a  review  of  the  entire  subject 
of  antibodies  and  immunity  in  general.  Vaughan  regards  it  as  a  ferment, 
evidently  meaning  that  an  amboceptor  and  a  complement  act  together 
and  produce  lysis  or  disruption  of  the  protein  molecule.  It  is  difficult, 
and  indeed  impossible,  at  this  time  to  discuss  with  any  degree  of  definite- 
ness  the  propriety  of  regarding  an  immune  and  cytolytic  amboceptor 
as  a  ferment,  just  as  we  regard  trypsin  as  a  ferment.  If  the  ferments 
are  to  be  considered  as  identical  with  amboceptors,  then  we  must  regard 
Abderhalden's  protective  ferments  as  cytolysins,  but  certainly,  in  the 
light  of  our  present  knowledge,  we  are  hardly  justified  in  drawing  hard 
and  fast  lines.  Therefore  while  I  have  confined  the  discussion  of  the 


ANAPHYLACTIN    (ALLERGIN)  589 

protective  ferments  to  a.  separate  chapter  (Chapter  XV) ,  and  have  not 
considered  it  under  the  general  head  of  the  cytolysins,  it  is  to  be 
remembered  that  the  ferments  possess  many  of  the  properties  of  lytic 
amboceptors,  and  they  may  be  identical  with  them  although  apparently 
different  owing  to  the  application  of  chemical  methods,  especially  by 
Abderhalden,  in  their  study. 

Most  observers  regard  the  anaphylactic  antibody,  toxogen  or  allergin, 
as  an  amboceptor  and  a  complement.  The  actual  antibody  then  must 
be  considered  as  an  immune  albuminolysin,  for  complement  is  present 
in  normal  serum,  and  is  not  necessarily  increased  during  the  process  of 
sensitization.  As  in  other  lytic  processes,  however,  complement  or 
alexin  is  of  great  importance,  constituting,  as  it  does,  the  actual  lytic 
agent,  after  the  antigen,  or,  in  this  instance,  the  anaphylactogen  of  the 
second  injection,  has  been  sensitized  by  the  amboceptor.  Some  ob- 
servers believe  that  the  complement  is  decreased  during  anaphylaxis, 
presumably  being  used  up  in  effecting  lysis  of  the  protein.  Friedmann 
claims  that  in  allergy  to  red  corpuscles  there  is  a  close  parallelism  between 
the  anaphylactic  bodies  and  the  hemolytic  amboceptor. 

With  the  more  recent  work  of  Abderhalden  on  protective  ferments 
and  the  development  of  a  dialysis  and  optical  method  of  detecting  the 
products  of  protein  cleavage,  it  was  hoped  that  the  true  and  exact  nature 
of  anaphylaxis  would  finally  be  established.  It  would  appear  possible 
to  determine  the  presence,  in  the  blood-serum  of  sensitized  animals,  of 
specific  proteolytic  ferments  capable  of  demonstrating  their  presence 
in  vitro,  and  to  show  the  products  of  protein  cleavage  just  after  anaphy- 
lactic shock  and  a  corresponding  decrease  or  total  absence  of  the  fer- 
ments as  a  result  of  their  participation  in  the  albuminolysis.  Indeed, 
Abderhalden1  has  recently  claimed  that  all  these  conditions  have  been 
found  to  exist:  (1)  The  serums  from  12  guinea-pigs  sensitized  to  egg- 
albumen,  when  mixed  with  antigen,  showed  digestive  power  by  both 
optic  and  dialysis  (biuret)  methods;  (2)  similar  serums,  dialyzed  alone, 
showed  digestive  products  in  only  one  of  six  serums  tested :  (3)  the  serum 
of  six  guinea-pigs  taken  at  intervals  of  from  five  minutes,  to  one  and  one- 
half  hours  after  the  second  injection  (egg-albumen)  and  dialyzed,  gave 
negative  results  after  from  five  and  fifteen  minutes,  whereas  four  taken 
after  thirty,  forty-five,  sixty,  and  ninety  minutes  respectively  were 
positive.  In  each  test  the  serum  (10  c.c.)  was  dialyzed  against  distilled 
water  for  sixteen  hours  at  37°  C.,  and  the  presence  of  the  products  of 
digestion  determined  by  the  biuret  reaction.  In  this  manner  the  final 
1  Zeitschr.  f.  physiol.  Chem.,  1912,  82,  109. 


590  ANAPHYLAXIS 

evidence  of  the  role  played  by  the  protein  split  products  in  the  produc- 
tion of  acute  anaphylaxis  has  apparently  been  furnished.  These  results 
however,  cannot  at  the  present  time  be  regarded  as  final.  Pearce  and 
Williams, 1  in  a  similar  study  with  horse  serum,  employing  the  dialysis 
technic  and  using  ninhydrin  as  the  indicator,  were  unable  to  demonstrate 
the  presence  of  the  ferments  after  sensitization,  although  by  using  large 
amounts  of  serum  secured  after  anaphylactic  shock  had  occurred  they 
observed  positive  reactions,  which  may  have  been  due,  in  part  at  least, 
to  the  presence  of  cleavage  products.  Zunz8  found  that  the  protein- 
splitting  properties  of  blood-serum,  as  tested  on  the  sensitizing  protein, 
increased  in  the  anaphylactic  state  from  the  fifth  to  the  twentieth  and 
sixtieth  day,  and  were  not  recognizable  in  blood-serum  taken  during 
or  soon  after  anaphylactic  shock  occurred.  Pfeiffer  and  Mita  and 
Vaughan  have  observed  the  apparent  disappearance  of  the  ferment  from 
the  blood,  even  though  the  animal  is  sensitized,  and  the  last-named 
observer  explains  this  on  the  assumption  that  the  ferment  comes  from 
the  fixed  cells,  which  are  stimulated  to  elaborate  the  ferment  only  when 
the  specific  protein  is  brought  into  contact  with  them. 

One  of  the  older  theories  of  anaphylaxis  regards  the  process  as  a 
precipitin  reaction.  It  is  apparent  that,  in  the  serum  of  an  animal 
immunized  with  a  soluble  protein,  such  as  a  serum,  a  precipitin  and  com- 
plement-fixing body,  presumably  an  albuminolysin  or  so-called  "  fer- 
ment, "  are  produced,  and  exist  together  in  the  immune  serum.  While 
the  role  of  precipitins  themselves  appear  to  have  been  excluded  as  directly 
participating  in  the  production  of  anaphylactic  shock,  recent  experi- 
ments of  Zinsser  and  others  would  tend  to  show  that  a  precipitin 
possesses  the  nature  of  a  protein  sensitizer  or  antibody  that  sensitizes 
its  antigen,  just  as  a  hemolytic  amboceptor  sensitizes  its  corpuscles, 
precipitation  being  a  secondary  phenomenon  due  to  the  colloidal  nature 
of  the  reacting  bodies  under  conditions  of  quantitative  proportions  and 
environment  that  favor  precipitation.  This  would  assign  to  the  pre- 
cipitins and  agglutinins  an  active  though  secondary  role  in  the  processes 
of  anaphylaxis  and  immunity. 

At  the  present  time,  therefore,  we  may  tentatively  assume  that  the 
anaphylactin  or  allergin  is  of  the  nature  of  a  specific  lytic  amboceptor 
or  albuminolysin,  which,  in  conjunction  with  non-specific  complement, 
constitutes  what  is  called  a  "ferment,"  and  is  capable  of  splitting  a 
protein  molecule  with  the  liberation  or  formation  of  a  toxic  moiety  re- 
sponsible for  the  lesions  and  symptoms  of  anaphylaxis.  Just  as  other 

1  Jour.  Infect.  Dis.,  1914,  14,  351.         2  Zeitschr.  f.  Immunitatsf.,  1913,  17,  241. 


THE    HUMORAL   THEORIES    OF   ANAPHYLAXIS  591 

normal  amboceptors,  such  as  hemolysins,  are  present  in  normal  serum, 
so  these  protein  amboceptors  or  albuminolysins  may  be  present  for 
various  proteins,  explaining  the  production  of  the  anaphylaxis  poison 
in  vitro  with  a  normal  serum  when  the  latter  is  fresh  and  active,  i.  e., 
when  complement  is  present. 

Theoretically,  at  least,  it  should  be  possible  to  detect  the  anaphy- 
lactic  amboceptor  in  a  serum  by  a  method  of  complement  fixation, 
although  practically  this  is  not  the  case.  The  whole  subject  of  " fer- 
ments" requires  further  study,  and,  as  a  result,  our  knowledge  and  views 
of  antibodies  and  the  processes  of  immunity  in  general  are  likely  to 
undergo  some  change. 

The  Humoral  Theories  of  Anaphylaxis. — With  the  foregoing  explana- 
tion of  the  chemical  or  humoral  theory  of  anaphylaxis,  I  may  briefly 
summarize  all  the  more  important  chemical  or  protein  poison  theories 
advanced  from  time  to  time  in  explanation  of  the  process.  These 
include  the  following: 

1.  Richet  held  that  the  sensitizer,  or  anaphylactogen,  contains  a 
substance  which  he  called  "congestion"  (because  he  did  his  original 
work  with  extracts  of  the  tentacles  of  sea  anemones,  which  are  toxic 
and  produce  congestion  of  the  internal  organs),  and  that  this  generates 
in  the  animal  another  substance,  known  as  the  "toxogenin."     The 
reaction  between  the  latter  and  the  homologous  protein  on  reinjection 
sets  free  a  poison,  "apo toxin,"  which,  because  of  its  effect  on  the  nervous 
system,  produces  the  symptoms  of  anaphylaxis.     This  theory  is  prac- 
tically the  same,  as  that  generally  accepted  today,  except  that  the  anti- 
gen is  not  of  necessity  primarily  toxic  for  the  animal. 

2.  Hamburger  and  Moro  suggest  that  the  first  injection  leads  to  the 
formation  of  precipitins,  and  that  on  reinjection  precipitates  are  formed; 
these  they  contend,  may,  by  the  formation  of  capillary  emboli,  produce 
acute  anaphylaxis,  or  atf  least  that  precipitin  formation  runs  parallel 
with  the  antibody  formation.    The  symptoms  of  anaphylaxis,  however, 
are  not  those  of  embolism,  and  there  is  no  evidence  to  show  that  pre- 
cipitation occurs  in  vivo,  although,  as  Zinsser  points  out,  precipitins  may 
play  the  role  of  sensitizers  of  the  antigen,  preparing  them  for  final  lysis 
or  cleavage  by  a  complement.    In  other  words,  the  precipitin  would  act 
as  an  amboceptor,  differing,  however,  from  our  general  conception  of 
the  nature  of  amboceptors  by  being  active  in  the  absence  of  complement, 
unless  precipitation  is  a  secondary  physical  phenomenon  in  the  nature 
of  a  colloidal  reaction. 

3.  Besredka  taught  that  the  sensitizer  contains  two  substances — 


592  ANAPHYLAXIS 

"sensibilisinogen"  and  "antisensibilisin."  When  injected  the  first 
time  the  former  develops  in  the  body  a  substance  called  "sensibilisin," 
and  on  reinjection  the  sensibilisin  and  antisensibilisin  continue  to  form 
a  poison  that  acts  on  the  nervous  system. 

4.  Gay  and  Southard  believe  that,  as  a  result  of  the  first  injection 
of  serum,  there  remains  in  the  circulation  a  protein  substance  called 
"anaphylactin,"  which  is  slowly  absorbed  and  continues  to  stimulate 
the  cells,  leading  to  an  abnormal  affinity  for  the  homologous  protein, 
which,  on  reinjection,  leads  to  anaphylactic  shock. 

5.  Vaughan  and  Wheeler  are  of  the  opinion  that,  with  the  parenteral 
introduction  of  a  foreign  protein,  the  body-cells  are  stimulated  to  produce 
a  specific  zymogen  or  ferment  that  digests  it.    The  protein  of  the  first 
injection  is  so  slowly  digested  that  the  effects  are  not  recognizable.    After 
the  protein  of  the  first  injection  has  been  disposed  of,  the  new  ferment 
continues  to  be  formed  in  the  cells,  and  on  the  second  injection,  after  the 
proper  interval  has  been  allowed  to  elapse,  this  zymogen  is  activated  and 
splits  up  the  protein,  which  promptly  and  abundantly  results  in  the  pro- 
duction of  the  symptoms  of  anaphylactic  shock.     Vaughan  believes 
that  there  is  a  non-specific  poisonous  group  or  moiety  in  each  protein 
molecule  which,  when  liberated  by  the  ferment,  is  responsible  for  ana- 
phylaxis.     This  poisonous  group  is  held  as  being  the  same  in  all  pro- 
teins, and  hence  the  similarity  of  lesions  and  symptoms  of  anaphylactic 
intoxication  in  animals  regardless  of  whether  the  protein  is  of  animal, 
vegetable,  or  bacterial  origin.     The  nature  of  the  ferment  is  not  clear. 
In  1907  they  regarded  it  as  a  zymogen — a  theoretic  labile  chemical  body 
resulting  from  intramolecular  rearrangement  in  the  protein  molecules 
of  the  cell.     Little  is  known  of  the  action  of  these  ferments  except  that 
in  some  manner  they  cause  cleavage  of  the  protein  molecule  and  libera- 
tion of  the  toxic  moiety.     Later,  Vaughan  speaks  of  the  ferment  as 
consisting  of  an  amboceptor  and  a  complement,  the  ferment   (pre- 
sumably the  complement  portion)  being  inactivated  by  a  temperature  of 
56°  C.  and  reactivated  on  the  addition  of  serum  and  organic  extracts. 
Although  Vaughan's  theory  best  explains  the  nature  and  source  of  the 
anaphylactic  poison,  that  of  Friedberger  explains  the  production  of  the 
"ferment,"  or  rather  the  protein  sensitizer  (amboceptor),  which,  with 
a  complement,  digests  the  protein  and  sets  free  or  produces  the  protein 
poison. 

6.  Friedberger  has  attempted  to  explain  anaphylaxis  on  the  basis 
of  Ehrlich's  side-chain  theory  of  the  action  of  antigens  and  the  produc- 
tion of  antibodies  similar  to  toxin-antitoxin  immunity.     This  theory 


THE   HUMORAL   THEORIES    OF   ANAPHYLAXIS  593 

assumes  that,  on  the  first  injection,  the  protein  finds  but  few  groups  of 
cellular  receptors  with  which  it  can  combine,  and  for  this  reason  it  is 
not  poisonous.  During  the  period  of  incubation  the  animal  cells  develop 
receptors  specific  for  the  homologous  protein;  with  a  single  small  dose 
of  protein  most  of  these  receptors  remain  attached  to  the  cell  (sessile) ; 
on  repeated  injections,  the  newly  formed  receptors  are  in  large  part  cast 
off  into  the  blood,  and  constitute  the  precipitins.  In  this  manner  an 
animal  relatively  insusceptible  to  a  foreign  protein  is  rendered  highly 
susceptible,  and  on  the  second  injection  the  protein  is  anchored  firmly 
to  the  cell,  just  as  the  cells  of  an  animal  may  anchor  diphtheria  toxin. 
One  of  the  essential  features  of  this  theory  is  that  it  assumes  that,  ordi- 
narily, the  receptors  are  not  preformed  in  sufficient  numbers  to  anchor 
enough  protein  to  injure  the  animal  with  the  first  injection,  regardless 
of  the  size  of  the  dose.  On  the  other  hand,  tetanus  and  diphtheria 
toxins  find  large  numbers  of  sessile  or  cellular  receptors,  and  are  highly 
toxic  on  the  first  injection.  As  originally  evolved,  the  theory  did  not 
explain  the  nature  of  the  toxic  agent  responsible  for  the  lesions  and  symp- 
toms of  anaphylaxis,  and  made  no  mention  of  the  protein  poison.  Never- 
theless it  affords  the  best  explanation  we  have  on  the  formation  of  the 
" ferment"  or  protein  sensitizer  (amboceptor).  At  one  time  Fried- 
berger  believed  that  anaphylaxis  could  be  explained  on  the  basis  of  a 
precipitin  reaction.  Anti-anaphylaxis  was  explained  on  the  assumption 
that  the  protein  of  the  reinjection  uses  up  the  sessile  receptors  already 
developed,  and,  accordingly,  not  enough  are  present  at  the  end  of  the 
period  of  incubation  to  produce  anaphylactic  shock.  Passive  anaphy- 
laxis was  explained  on  the  ground  that  the  free  receptors  in  the  blood 
of  a  sensitized  animal  become,  on  injection  into  a  fresh  animal,  anchored 
to  the  cells,  thus  forming  fixed  or  sessile  receptors  that  anchor  the  pro- 
tein on  reinjection  and  lead  to  anaphylaxis. 

Nolf1  has  proposed  a  theory  of  anaphylaxis  that  has  come  to  be 
known  as  the  ''physical  theory."  It  assumes  that  the  active  constituent 
of  proteins  is  a  thromboplastic  substance  that  disturbs  the  colloidal 
equilibrium  of  the  blood  and  leads  to  the  deposition,  on  the  surface  of 
the  leukocytes  and  the  endothelial  cells  of  capillaries,  of  a  delicate  film 
of  fibrin.  Thus  stimulated,  the  cells  pour  out  an  unusual  amount  of 
antithrombin.  On  account  of  the  consumption  of  a  part  of  the  fibrino- 
gen  and  the  increased  formation  of  antithrombin  the  blood  fails  to  coagu- 
late after  anaphylactic  shock  or  peptone  poisoning.  Owing  to  the 
coagulation  deposits  on  the  endothelial  cells,  the  viscidity  is  increased 

1  Quoted  by  Vaughan,  "Protein  Split  Products,"  1913,  340. 

38 


594  ANAPHYLAXIS 

and  the  leukocytes  adhere  to  the  vessel-walls,  thus  accounting  for  the 
leukopenia  observed  after  protein  injection.  The  enclothelial  cells  are 
injured,  and  the  walls  of  the  capillaries  become  more  readily  permeable, 
thus  accounting  for  the  local  edema  often  seen  in  anaphylaxis.  The 
fine  capillaries  of  a  given  area  may  be  occluded  by  thrombi,  thus  ex- 
plaining the  necrosis  characteristic  of  the'Arthus  phenomenon.  The 
irritation  of  the  endothelial  cells  extends  to  the  smooth  muscle,  leading 
to  vasoparalysis  and  the  characteristic  fall  in  blood-pressure.  The 
affinity  of  the  endothelial  cells  for  the  protein  is  stimulated  by  the  first 
injection,  and  acts  in  a  fulminating  way  on  reinjection,  thus  explaining 
the  suddenness  of  anaphylactic  shock. 

The  theory,  therefore,  also  assumes  the  formation  of  a  ferment  that 
acts  primarily  upon  the  proteins  of  the  blood,  leading  to  the  formation 
of  fibrin,  which,  as  it  were,  mechanically  induces  the  lesions  and  symptoms 
of  anaphylaxis.  While  it  offers  a  plausible  explanation,  the  theory  is 
not  well  supported,  and  at  best  may  be  regarded  as  a  modification  of 
Vaughan's  theory,  demonstrating  one  way  in  which  the  protein  poison 
may  act. 

THE  CELLULAR  THEORY  OF  ANAPHYLAXIS 

Many  investigators  have  attempted  to  explain  anaphylaxis  on  the 
basis  that  the  reaction  is  a  cellular  one,  that  is,  that  the  antibody  is 
within  the  cell  and  that  the  antigen-antibody  reaction  occurs  in  this 
position  rather  than  in  the  blood-stream  by  means  of  free  or  circulating 
antibody  and  antigen.  According  to  the  "cellular  theory,"  if  the  serum  of 
an  immunized  animal  containing  the  anaphylactic  antibody  is  injected 
into  a  normal  animal  (passive  anaphylaxis)  and  is  followed  by  an  injec- 
tion of  the  antigen,  an  anaphylactic  reaction  cannot  occur  before  the 
elapse  of  sufficient  time  for  the  antibody  to  become  anchored  to  cells. 
The  "humoral  theory,"  on  the  other  hand,  assumes  that  the  antigen 
meets  the  antibody  in  the  blood-stream  and  explains  the  time  required 
between  the  injection  of  immune  serum  and  antigen  in  passive  anaphy- 
laxis as  due  to  a  failure  of  rapid  union  between  antigen  and  antibody 
unless  qualitative  relations  between  the  two  are  accidentally  correct. 

The  early  theory  of  Friedberger,1  explaining  anaphylaxis  on  the  basis 
of  "sessile  receptors";  the  experiments  of  Friedberger  and  Girgolaff,2 
who  passively  sensitized  normal  animals  by  transplanting  the  thoroughly 
washed  organs  of  a  sensitized  animal;  the  transfusion  experiments  of 

1  Ztsch.  f.  Immunitatsf.,  orig.,  1909,  11,  208. 

2  Ztsch.  f.  Immunitatsf.,  orig.,  1911,  ix,  575. 


THE    CELLULAR    THEORY    OF    ANAPHYLAXIS  595 

Pearce  and  Eisenbrey,1  who  transferred  the  blood  of  a  sensitized  animal 
to  a  normal  animal  and  the  blood  of  a  normal  animal  to  a  sensitized  one, 
finding  that  the  latter,  but  not  the  former,  reacted  when  the  antigen 
was  injected  as  soon  as  the  transfusions  were  completed;  the  work 
of  Coca,2  who  found  that  sensitized  guinea-pigs  would  still  react  after 
being  thoroughly  bled  and  perfused  with  salt  solution ;  the  investigations 
of  Schultz,3  Dale,4  and  particularly  of  Weil,5  showing  that  the  excised 
and  washed  muscles  of  sensitized  animals  would  react  in  vitro  in  a  bath 
of  Ringer's  solution  when  the  antigen  was  added,  support  most  strongly 
the  cellular  theory  of  anaphylaxis  as  emphasized  also  in  the  work  and 
recent  communications  of  Doerr.6 

In  the  opinion  of  Weil,  Doerr,  Bayliss,  Coca,  and  others  the  "cellular" 
theory  is  the  only  tenable  one  today.  According  to  this  theory  of  ana- 
phylaxis, the  antibody  is  in  or  on  the  body  cells;  upon  union  with  the 
antigen  the  cells  undergo  a  physical  shock  which  has  been  likened  to 
an  electric  shock,  and  this  constitutes  the  basis  of  the  anaphylactic 
reaction  without  the  formation  of  any  intermediate  or  chemical  poison. 

As  emphasized  by  Weil7  there  is  no  direct  evidence  of  the  production 
of  a  chemical  poison  or  anaphylatoxin  during  or  after  an  anaphylactic 
reaction  in  the  living  animal.  This  poison  has  not  been  satisfactorily 
demonstrated  in  the  blood  and  in  animals  recovering  from  a  general  ana- 
phylactic reaction,  the  phenomena  being  more  suggestive  of  a  transitory 
"shock"  than  of  an  intoxication  with  a  chemical  poison. 

On  the  other  hand,  the  mass  of  experimental  evidence  indicating 
that  the  anaphylactic  reaction  is  not  purely  cellular,  but  at  least  in  part 
an  intra vascular  reaction,  is  too  strong  to  be  discarded.  As  pointed  out 
by  Zinsser  and  Young,8  the  necessary  time  usually  required  in  passive 
anaphylaxis  between  the  injection  of  immune  serum  and  antigen  may 
be  due  to  slow  union  between  antigen  and  antibody  in  the  blood-stream, 
on  the  basis  that  the  anaphylactic  reaction  involves  the  interaction  of 
colloids  and  that  a  protective  colloid  is  responsible  for  the  slow  union 
of  antigen  and  antibody  unless  its  influence  is  obviated  by  exact  quanti- 
tative proportions  between  antigen  and  antibody,  which  under  experi- 
mental conditions  may  be  secured  in  a  more  or  less  accidental  manner. 

1  Congr.  Amer.  Phys.  and  Surg.,  1910,  viii,  402. 

2  Ztsch.  f.  Immunitatsf.,  orig.,  1914,  xx,  622. 

3  Jour.  Pharmacol.  and  Exper.  Therap.,  1910,  1,  549. 

4  Jour.  Pharmacol.  and  Exper.  Therap.,  1913,  iv,  167. 

5  Jour.  Med.  Research,  1914,  xxx,  87. 

'  6  Ergebnisse  der  Immunitatsf.,  Reichhardt,  Berlin,  1914,  1,  257. 

7  Proc.  Soc.  Exper.  Biol.  and  Med.,  1915,  xiii,  23  (1087). 

8  Jour.  Exper.  Med.,  1913,  xvii,  396. 


596  ANAPHYLAXIS 

As  antibodies  are  produced  by  the  body  cells,  it  is  reasonable  to 
believe  that  they  are  present  in  the  protoplasm  of  the  cells,  and,  even 
after  thorough  washing  of  the  tissues,  are  capable  of  union  with  their 
antigen.  It  is  entirely  likely  that  these  attached  or  sessile  antibodies 
are  chiefly  concerned  in  the  phenomena  of  anaphylaxis,  but  I  cannot 
subscribe  entirely  to  the  cellular  theory,  because  of  the  mass  of  experi- 
mental data  bearing  on  the  production  of  the  protein  poison,  particu- 
larly in  vitro,  by  the  free  antibodies  in  an  immune  serum.  The  reactions 
in  vivo  of  hemolysis  and  bacteriolysis,  which  are  lytic  processes  appar- 
ently similar  to  those  concerned  in  anaphylactic  reactions,  are  probably 
intravascular  processes;  it  is  also  likely  that  in  anaphylaxis  to  formed 
antigens  the  first  phase  of  the  reaction,  that  is,  the  disruption  of  the 
antigenic  cell,  is  intravascular,  showing  that  lytic  reactions  entirely 
analogous  to  our  conception  of  the  mechanism  of  the  production  of  the 
protein  poison  in  anaphylactic  reactions  may  occur  in  the  blood-stream. 
For  these  reasons  alone  I  cannot  exclude  the  free  antibodies  in  the  blood 
from  playing  some  role  in  the  phenomena  of  anaphylaxis. 

The  extreme  sensitiveness  of  the  body  cells  in  anaphylaxis  to  the 
protein  suggests  to  me  that  the  cells  acquire  the  property  of  union  with 
the  protein  poison  to  an  extreme  degree,  as  if  they  were  furnished,  as  a 
result  of  the  initial  dose  of  foreign  protein,  with  an  increased  number 
of  specific  and  sessile  receptors  for  the  protein  poison.  In  other  words, 
while  the  protein  poison  may  be  produced  in  the  cells  by  sessile  recep- 
tors or  in  the  blood-stream  by  free  receptors,  anaphylaxis  itself,  that  is, 
the  hypersensitiveness  of  the  cells,  is  due  to  the  increased  binding  power 
of  the  cells  for  the  protein  poison.  In  our  opinion  this  in  part  explains 
true  allergy,  that  is,  the  effects  of  local  or  general  sensitization  (pro- 
duction in  the  protoplasm  of  the  cells  or  increased  receptors  for  the 
protein  poison),  and  the  immediate  well-marked  or  even  violent  effects 
following  the  production  of  what  must  be  very  minute  amounts  of  protein 
poison,  as  in  the  cutaneous  tuberculin  reaction  or  in  the  classical  horse- 
serum  reaction  in  guinea-pigs,  following  the  intravenous  injection  of 
the  intoxicating  or  second  dose  of  protein  in  very  minute  or  infini- 
tesimal dosage. 

PASSIVE  ANAPHYLAXIS 

Passive  anaphylaxis  is  produced  by  the  injection  of  normal  animals 
with  the  blood  or  serum  of  animals  or  persons  already  sensitized.  It 
is  similar  to  passive  immunization,  and  is  specific  for  the  anaphylactogen 
with  which  the  donor  is  sensitized. 


PASSIVE    ANAPHYLAXIS  597 

The  second  animal  may  be  of  the  same  or  of  another  species.  If 
it  is  of  the  same  species,  the  condition  induced  by  the  transference  of 
the  serum  is  called  homologous;  if  it  is  of  a  different  species,  it  is  known 
as  heterologous,  or  passive  anaphylaxis. 

Passive  anaphylaxis  was  discovered  almost  simultaneously  and  in- 
dependently by  Gay  and  Southard,1  working  with  guinea-pigs,  by 
Nicolle,2  with  rabbits,  and  by  Otto,3  who,  working  with  both  guinea-pigs 
and  rabbits,  showed  that  a  rabbit  immune  serum  would  passively  sensi- 
tize a  guinea-pig. 

At  this  place  it  may  be  mentioned  that  the  new-born  of  a  sensitized 
mother  animal  may  be  sensitive,  and  remain  so  for  a  longer  or  shorter 
period  of  time.  This  is  an  illustration  of  passive  homologous  anaphy- 
laxis, a  process  that  has  been  especially  studied  by  Rosenau  and  Anderson, 
Gay  and  Southard,  and  Otto,  the  last  observer  finding  that  young  guinea- 
pigs  remained  sensitive  for  as  long  as  forty-five  days  after  birth.  This 
function  of  transmitting  the  condition  of  sensitization  is  solely  maternal: 
the  male  takes  no  part  whatever  in  the  transmission  of  these  acquired 
properties. 

The  Production  of  Passive  Anaphylaxis. — An  important  phase  of 
this  subject  over  which  there  has  been  considerable  difference  of  opinion 
refers  to  the  question  whether  some  time  must  elapse  between  the  in- 
jection of  immune  serum  and  anaphylactogen  before  anaphylaxis  is 
produced,  or  whether  intoxication  may  follow  the  simultaneous  injection 
of  both  antibody  and  antigen.  Thus  Gay  and  Southard,  in  their  early 
studies,  found  their  recipients  first  sensitized  on  the  fourteenth  day 
after  injection  of  immune  serum.  Otto  and  Friedmann  observed  shock 
twenty-four  hours  after  injecting  the  anaphy lactic  serum  subcutaneously 
and  antigen  intraperitoneally.  By  injecting  both  serums  intravenously 
and  simultaneously,  Doerr  and  Russ  finally  succeeded  in  producing  acute 
anaphylaxis  and  almost  immediate  death.  Weil,  ^however,  believes  that 
the  simultaneous  injection  of  antigen  and  of  antiserum  into  opposite 
jugular  veins  in  the  guinea-pig  never  produces  an  anaphylactic  reaction, 
in  spite  of  the  use  of  wide  quantitative  variations  in  both  substances. 
On  the  other  hand,  if  the  antigen  were  injected  a  few  hours  after  the 
antiserum,  in  the  same  quantitative  variations,  the  reaction  occurred 
regularly.  From  this  it  was  concluded  that  the  body-cells  anchor  the 

1  Jour.  Med.  Research,  1907,  xvi,  143. 

2  Ann.  de  Flnst.  Pasteur,  1907,  xxi,  128. 

3  Munch,  med.  Wchnschr.,  1907,  No.  39. 

4  Jour.  Med.  Research,  1914,  30,  No.  2,  87. 


598  ANAPHYLAXIS 

antibody  during  the  latent  period,  and  that  anaphylaxis  is  the  result 
of  an  interaction  between  the  cellular  antibodies  and  the  antigen. 

It  would  appear  that  passive  anaphylaxis  is  not  wholly  determined 
by  the  amounts  of  antigen  or  antibody,  but  by  the  proportion  that 
exists  between  the  two.  For  example,  Friedmann,1  in  his  studies  on 
passive  homologous  anaphylaxis  in  rabbits,  found  that,  by  employing 
2.5  c.c.  of  antiserum  with  from  2.5  to  0.25  c.c.  of  antigen,  no  results  were 
observed,  whereas  positive  reactions  were  obtained  when  the  amount  of 
antigen  was  reduced  from  0.025  to  0.0025  c.c. 

Ordinarily,  normal  guinea-pigs  may  be  passively  sensitized  by  0.1  to 
0.5  c.c.  of  serum  injected  intraperitoneally,  and  anaphylactized  one  or 
two  days  later  by  an  intravenous  injection  (0.1  to  0.5  c.c.)  of  the  antigen. 
The  immune  serum  may  be  prepared  by  injecting  rabbits  with  horse 
serum,  after  the  methods  for  the  production  of  precipitins  described  in 
Chapter  IV. 

The  duration  of  passive  sensitization  is  quite  variable.  Weil2  found 
that  guinea-pigs  sensitized  with  a  homologous  serum,  that  is,  with  the 
serum  of  another  guinea-pig  sensitized  with  horse  serum,  remain  typic- 
ally anaphylactic  as  long  as  seventy  days  after  the  injection.  With 
heterologous  sensitization,  however,  as  with  the  serum  of  a  sensitized 
rabbit,  hypersensitiveness  is  almost  invariably  lost  by  the  tenth  day. 
One  explanation  of  this  would  be  that  a  heterologous  serum  is  excreted 
more  rapidly  than  a  homologous  serum,  a  condition  commonly  observed 
in  serum  therapy  with  any  heterologous  serum. 

The  Mechanism  of  Passive  Anaphylaxis. — Presumably  the  mecha- 
nism of  passive  anaphylaxis  in  terms  of  the  humoral  theory  is  relatively 
simple,  and  consists  in  the  transfer  of  the  specific  protein  sensitizer  or 
amboceptor  that  unites  the  anaphylactogen  or  protein  antigen  with  a 
complement,  bringing  about  lysis  or  cleavage  of  the  protein  and  liber- 
ation of  the  toxic  moiety  responsible  for  the  lesions  and  symptoms  of 
anaphylaxis.  In  other  words,  the  mechanism  of  passive  anaphylaxis 
may  be  likened  to  passive  antibacterial  immunization,  with,  however, 
one  important  clinical  difference,  namely,  that  whereas  in  the  former 
the  microorganisms  are  destroyed  without  apparent  injury  to  the 
host,  in  anaphylaxis  the  body-cells  are  acutely  poisoned.  However,  a 
similar  phenomenon  in  the  serum  treatment  of  disease,  with  lysis  of 
bacteria,  may  be  overshadowed  or  possibly  prevented  by  a  condition 
of  anti-anaphylaxis. 

According  to  the  cellular  theory,  passive  anaphylaxis  is  due  to  the 

v    «' 

1  Jahr.  u.  d.  Ergeb.  Immunitatsf.,  1910,  vi,  67. 

2  Jour.  Med.  Research,  1913,  28,  No.  2,  359. 


ANTI-ANAPHYLAXIS    OR    DESENSITIZATION  599 

anchoring  of  the  anaphylactic  antibody  to  the  body-cells,  and,  according 
to  Weil,  sufficient  time  must  always  elapse  after  the  injection  of  serum 
for  this  union  to  occur. 

ANTI-ANAPHYLAXIS  OR  DESENSITIZATION 

The  term  anti-anaphylaxis  was  first  applied  by  Besredka  and  Stein- 
hardt  to  a  condition  of  insensibility  to  further  injection  of  the  anaphylac- 
togen  that  may  follow  recovery  from  anaphylaxis,  or  be  induced  arti- 
ficially by  a  single  or  by  repeated  small  injections  of  the  anaphylactogeh 
during  the  incubation  period  following  the  first  injection,  and  before 
sensitization  is  completed.  The  state  is  usually  only  temporary,  the 
animal  gradually  becomes  sensitive  again  after  three  weeks. 

Theobald  Smith  had  observed  that  those  guinea-pigs  that  had  re- 
ceived the  largest  dose  of  diphtheria  toxin-antitoxin  mixture  more 
frequently  survived  the  second  dose  than  did  those  that  received  smaller 
doses.  Rosenau  and  Anderson  found  that  animals  reinjected  before 
the  end  of  the  period  of  incubation  did  not  become  responsive  until 
some  time  later.  Otto  has  also  made  this  observation,  but  the  most 
thorough  study  of  the  subject  has  come  as  the  result  of  the  researches  of 
Besredka  and  Steinhardt. 

Experimental  Production  of  Anti-anaphylaxis. — It  has  long  been 
known  that  the  larger  the  first  or  sensitizing  injection  of  antigen,  the 
greater  must  be  the  dose  of  the  second  or  intoxicating  injection,  indicat- 
ing that  a  large  sensitizing  injection  introduces  the  factor  tending  to 
produce  the  condition  of  anti-anaphylaxis.  Partial  desensitization, 
or  anti-anaphylaxis,  may  be  produced  by  the  injection  of  a  sublethai 
intoxicating  dose  of  anaphylactogen  during  the  period  of  incubation  or 
at  its  close.  Quantitative  relations  between  the  size  of  the  sensitizing, 
intoxicating,  and  desensitizing  doses  of  antigen  and  the  period  of  in- 
cubation have  been  worked  out  in  a  series  of  studies  by  Weil,1  both  in 
the  living  guinea-pig  and  on  the  excised  uterus,  after  the  graphic  method 
of  Dale.2  Weil  found  that  a  small  sensitizing  dose  of  horse  serum  (0.01 
c.c.  subcutaneously)  is  followed  by  a  relatively  prolonged  period  of 
incubation  (from  fourteen  to  sixteen  days) ;  that  the  minimal  anaphy- 
lactic or  lethal  dose  is  small  (0.02  to  0.05  c.c.) ;  that  the  blood,  as  a  rule, 
does  not  contain  more  than  one  sensitizing  unit;  and  that  the  minimal 
desensitizing  dose  is  small  (0.01  c.c.).  Conversely,  after  repeated 
large  sensitizing  doses  (2  c.c.  of  serum  subcutaneously  on  each  of  three 

1  Jour.  Med.  Research,  1913,  29,  No.  2,  233;  1914,  30,  No.  3,  299. 

2  Jour.  Pharm.  and  Exper.  Ther.,  1913,  iv,  167. 


600  ANAPHYLAXIS 

days  in  succession)  the  incubation  period  is  shorter  (about  ten  days 
after  the  last  injection);  the  minimal  anaphylactic  lethal  dose  is  larger 
(0.4  c.c.),  and  the  minimal  desensitizing  dose  is  at  least  more  than  0.2 
c.c.  of  serum  given  intravenously. 

Besredka  and  Steinhardt  observed  that  the  refractory  state  could 
easily  be  developed  in  sensitized  guinea-pigs  by  one  of  the  following 
methods:  (1)  The  intracerebral  injection  of  0.25  c.c.  of  horse  serum 
before  the  expiration  of  the  period  of  incubation  (twelve  days).  (2)  The 
intracerebral  injection  of  less  than  the  fatal  dose  (from  0.002  to  0.025  c.c.) 
after  the  period  of  incubation.  (3)  Rectal  injections  of  from  5  to  10 
c.c.  of  serum.  (4)  By  slowly  reinjecting  small  amounts  while  the 
animal  is  deeply  narcotized  with  ether  or  alcohol.  As  shown  later  by 
Rosenau  and  Anderson,  a  narcotic  may  mask  but  does  not  prevent  the 
occurrence  of  severe  or  fatal  symptoms.  Of  these  methods,  Besredka 
prefers  the  rectal  injection,  or,  better  still,  the  subcutaneous  injection 
of  less  than  a  fatal  dose. 

The  subject  of  anti-anaphylaxis  is  of  great  importance  from  its  rela- 
tion to  serum  therapy.  No  satisfactory  method  for  producing  this 
state  in  a  sensitized  person  has  as  yet  been  devised,  owing,  probably,  to 
the  important  quantitative  factors  shown,  by  the  studies  of  Weil,  to  exist. 
This  subject  will  be  discussed  again  in  the  following  chapter,  under  the 
head  of  Serum  Sickness. 

The  Mechanism  of  Anti-anaphylaxis. — A  true  explanation  of  this 
phenomenon  cannot  as  yet  be  given.  In  the  first  place,  the  term  anti- 
anaphylaxis  cannot  be  considered  a  proper  one,  as  the  animal  is  not 
entirely  and  permanently  anti-anaphylactic,  but  subsequently  becomes 
sensitive.  The  blood-serum  of  a  refractory  or  anti-anaphylactic  animal 
does  not  confer  a  similar  condition  on  a  second  sensitized  animal. 

As  previously  stated,  Friedberger  believes  that  the  refractory  state 
is  due  to  neutralization  or  absorption  of  the  anaphylactic  antibody  by 
the  antigen,  but  this  explanation  does  not  fit  in  with  the  facts,  first, 
because  the  serum  of  an  anti-anaphylactic  animal  will  still  passively 
sensitize  a  normal  animal,  and,  secondly,  as  shown  by  Weil,  passive 
anaphylaxis  of  a  guinea-pig,  such  as  that  induced  by  the  injection  of  a 
rabbit  antihorse  serum,  may  be  prevented  for  at  least  eight  days  by  a 
previous  injection  of  normal  rabbit  or  sheep  serum.  In  other  words, 
it  would  appear  that  normal  rabbit  and  sheep  serum  may  protect  the 
body-cells  of  the  guinea-pig  against  the  anaphylatoxin  produced  by 
horse  protein  and  horse  anaphylactin  or  antibody.  In  explanation  of 
this  paradoxic  and  non-specific  reaction  Weil,1  who  believes  in  the  cel- 
1  Jour.  Med.  Research,  1914,  30,  No.  3,  299. 


SPECIFICITY   OF   ANAPHYLAXIS  601 

lular  theory  of  anaphylaxis,  has  tentatively  advanced  the  hypothesis 
that  the  indifferent  serum  persists  in  the  body-cells  and  markedly  lowers 
the  reactivity  of  the  cellular  antibodies. 


SPECIFICITY  OF  ANAPHYLAXIS 

The  anaphylactin,  or  so-called  anaphylaxis  antibody,  displays  quite 
the  same  characteristics  of  specificity  as  do  the  other  immune  anti- 
bodies, in  that  proteins  of  closely  related  species  tend  to  interact,  whereas 
proteins  of  very  distinct  biologic  or  chemical  nature  are  easily  distin- 
guished. In  other  words,  the  anaphylactic  reaction  is  highly  specific,  and 
of  considerable  value  in  the  study  and  identification  of  different  proteins. 
For  example,  Dale  has  recommended  the  use  of  the  graphic  method  in 
ntro  with  excised  muscle  (uterus)  for  the  identification  of  the  protein 
substances,  such  as  blood-stains.  Guinea-pigs  sensitized  with  human 
serum  will  react  best  with  human  serum,  and  to  a  lesser  extent  with 
that  of  the  higher  apes,  but  not  at  all  with  the  serum  of  the  dog,  ox,  or 
fowl.  Wells  and  Osborne,1  working  with  purified  vegetable  protein, 
were  able  to  demonstrate  that  a  single  isolated  protein  (hordein  or 
gliadin)  may  contain  more  than  one  antigenic  radical,  and  that  not  the 
whole  protein  molecule,  but  certain  groups  thereof,  determine  the 
specificity.  Wells2  was  able  to  distinguish  in  the  hen's  egg  five  dis- 
tinctly different  antigens,  and  these  corresponded  to  five  proteins  that 
were  isolated  by  chemical  methods,  so  that  it  would  appear  that  the 
specificity  of  the  anaphylaxis  reaction  is  determined  by  the  chemical  struc- 
ture of  the  reacting  proteins,  rather  than  by  their  biologic  origin.  Whether 
the  chemical  differences  that  determine  specificity  are  of  quantitative 
nature,  or  whether  they  are  sometimes  dependent  upon  the  number  and 
relationship  of  the  amino-radicals,  as  was  suggested  by  Pick,  remains 
to  be  determined. 

1  Jour.  Infect.  Dis.,  1913,  12,  341.  2  Jour.  Infect.  Dis.,  1911,  9,  147. 


PART  IV 

CHAPTER  XXVIII 

ANAPHYLAXIS  IN  ITS  RELATION  TO  INFECTION  AND 

IMMUNITY 

IN  reviewing  our  knowledge  of  the  nature  and  mechanism  of  anaphy- 
laxis  we  found  that  ordinarily  innocent  substances,  such  as  sterile  normal 
serum  and  egg-albumen,  when  injected  into  animals,  may  give  rise  to 
severe  and  even  fatal  intoxication,  not  because  these  substances  are 
poisonous  in  themselves,  but  because  the  antibodies  which  they  stimu- 
late the  body-cells  to  produce  react  with  the  innocent  protein  in  the 
body  fluids  or  cells  or  both  producing  the  phenomenon  called  anaphy- 
laxis.  Here,  indeed,  we  have  an  example  of  antibodies  apparently 
injuring  our  body-cells  instead  of  protecting  them.  And  now  the  ques- 
tion very  naturally  arises,  are  the  lesions  of  the  many  diseases  caused 
in  a  similar  manner,  the  antibodies  acting  with  the  bacterial  cell  and 
producing  the  anaphylactic  state?  In  view  of  the  fact  that  they  may  do 
actual  harm,  in  some  instances  at  least,  are  antibodies  really  protective 
and  beneficial,  and  if  so,  in  what  manner? 

It  is  not  my  purpose  to  review  here  the  general  subjects  of  infection 
and  immunity,  but  to  discuss  briefly  the  intimate  connection  of  what 
we  call  anaphylaxis  or  allergy  with  infection,  and  to  show  that  anaphy- 
laxis  may  be  the  first  step  in  the  process  of  immunity;  indeed,  that  well- 
marked  antibacterial  immunity  may  be  an  example  of  an  early  and 
efficient  "anaphylactic"  reaction.  In  other  words,  while  the  striking 
and  severe  symptoms  of  serum  anaphylaxis  in  either  man  or  lower 
animals  give  us  the  impression  that  we  are  here  dealing  with  some  new, 
distinct,  and  strange  phenomenon,  these  may  be,  in  fact,  but  exaggerated 
and  severe  examples,  largely  dependent  upon  quantitative  factors  of 
what  occurs  during  each  infection.  More  than  this,  in  the  terms  of  the 
humoral  theory  of  anaphylaxis  these  symptoms  may  be  due  in  part 
to  the  action  of  a  poison  formed  or  released  through  the  destruction  of 
the  antigen  by  the  antibody;  the  antibody  being,  therefore,  apparently 
imperfect  in  its  action,  as  it  does  not  neutralize  the  protein  poison. 
Taking,  as  an  example,  a  substance,  such  as  sterile  horse  serum,  while 

ordinarily  harmless  itself,  bad  effects  may  follow  its  injection  at  once 

602 


RELATION    OF   ANAPHYLAXIS   TO    INFECTIOUS    DISEASES  603 

if  antibodies  are  present  in  our  body-fluids,  or  later  if  some  of  the  serum 
persists  in  the  body  until  antibodies  are  produced.  It  would  appear, 
therefore,  that  when  the  antibodies  are  produced  for  a  given  infectious 
agent,  then  the  severity  of  the  disease  becomes  apparent ;  that  the  lesions 
and  symptoms  are  due  not  only  to  the  infecting  microorganism  and  its 
products,  but  to  the  results  of  their  destruction.  An  invading  army 
may  do  some  pillaging,  but  the  greatest  injury  is  done  when  the  de- 
fenders begin  the  attack,  the  resulting  fire  and  destruction  doing  more 
harm  than  the  invaders  themselves. 

While  Vaughan  and  his  coworkers  were  studying  the  protein  poison 
in  vitro,  and  Friedberger  was  investigating  it  in  vivo,  the  former  and  then 
the  latter  aiming  to  show  that  the  poison  liberated  from  the  protein 
molecule  through  the  action  of  specific  ferments  is  responsible  for  the 
lesions  and  symptoms  of  disease,  von  Pirquet  was  studying  the  question 
from  the  clinical  aspect,  formulating  a  working  hypothesis  on  the  nature 
of  infection  and  immunity  based  upon  the  principles  of  anaphylaxis,  a 
theory  that  has  been  supported  by  experimental  data  and  has  thrown  a 
new  light  upon  the  nature  and  mechanism  of  these  processes. 


RELATION  OF  ANAPHYLAXIS  TO  INFECTIOUS  DISEASES 
Although  we  may  not  be  in  general  accord  regarding  the  mechanism 
of  anaphylaxis,  if  the  humoral  theory  is  accepted  there  is  a  general 
agreement  as  regards  the  nature  of  the  anaphylatoxin  responsible  for 
the  lesions  and  symptoms  of  anaphylactic  intoxication,  namely,  that 
it  is  a  protein  cleavage  product.  In  other  words,  a  foreign  protein  and, 
indeed,  the  misplaced  protein  of  our  own  body-cells  may  be  disrupted 
or  digested  by  an  antibody,  and  liberate  or  generate  a  poison  that, 
being  derived  from  protein,  is  known  as  the  protein  poison.  In  the 
chapter  on  Infection  it  was  stated  that  Vaughan  and  his  collaborators 
regard  this  protein  poison  as  the  same  for  all  proteins,  and  as  responsible 
for  all  infectious  diseases,  the  particular  lesions  and  symptoms  of  each 
disease  being  dependent  upon  the  site  of  the  infection  and,  accordingly, 
upon  the  location  of  the  protein  poison.  Similarly,  in  the  preceding 
chapter  we  asserted  that  anaphylaxis  may  be  ascribed  to  an  exactly 
similar  phenomenon,  namely,  the  splitting  of  the  foreign  protein  by  an 
antibody  (ferment)  and  the  liberation  of  a  protein  poison.  In  studying 
serum  sickness,  an  anaphylactic  phenomenon  frequently  observed  in 
man  following  the  administration  of  horse  serum,  von  Pirquet  argued 
that  the  period  of  from  eight  to  ten  days  usually  following  the  injection 


604      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

before  the  appearance  of  symptoms  was  the  time  required  for  the  produc- 
tion of  the  antibody,  which  then  reacted  upon  the  serum  still  remaining 
in  the  body-cells  and  fluids,  and  that  the  products  of  this  interaction 
caused  the  lesions  and  symptoms  of  serum  sickness.  It  was  then  but 
a  short  step  to  apply  these  principles  to  other  infectious  diseases.  This 
"period  of  incubation"  was  formerly  regarded  as  representing  a  stage 
during  which  the  infecting  microorganisms  multiply  in  the  body  of  the  in- 
fected individual,  to  that  point  at  which  they  could  give  rise  to  symp- 
toms of  disease  through  the  agency  of  their  toxins  or  through  interference 
with  the  metabolism  of  the  host  in  other  ways.  But,  as  von  Pirquet  has 
pointed  out,  this  theory  does  not  hold  in  serum  sickness,  as  the  serum  may 
be  sterile  arid  no  infecting  microorganisms  are  at  work.  Instead,  he  and 
Vaughan  would  have  us  believe  that  during  this  period  antibody  forma- 
tion is  taking  place,  and  that  an  antibody-antigen  reaction  will  occur  with 
the  development  of  pathologic  changes  and  symptoms  just  as  soon  as 
these  changes  have  progressed  to  a  certain  point.  The  period  of  incuba- 
tion will  vary  not  only  in  point  of  time  of  reaction,  but  also  qualitatively 
and  quantitatively,  and  using  this  as  a  basis  von  Pirquet  recognizes 
three  main  groups,  depending  upon  whether  the  antibody  is  present  in 
our  body-fluids  as  the  result  of  a  previously  acquired  infection  (acci- 
dental or  by  vaccination),  or  whether  it  must  first  be  developed. 

Group  I :  Reaction  appears  after  eight  to  twelve  days,  as  in  measles, 
smallpox,  whooping-cough,  chickenpox,  and  other  infectious  diseases  in 
which  the  antibodies  must  be  developed  before  the  symptoms  are  pro- 
duced. This  interval  corresponds  quite  closely  to  that  observed  in 
serum  sickness.  If  at  this  time  the  antigen, — i.  e.,  either  the  albumins 
of  the  horse  serum,  if  we  are  dealing  with  serum  injections,  or  the  bac- 
teria in  case  of  an  infection — has  disappeared  from  the  body,  no  symptom 
will,  of  course,  result;  if,  however,  some  of  the  material  is  still  present, 
a  reaction  occurs,  during  which  the  protein  poison  (anaphylatoxin)  is 
produced,  and  to  which,  in  turn,  the  symptoms  that  then  develop  may 
logically  be  attributed. 

Group  II:  The  reaction  appears  after  three  to  seven  days.  If,  on 
the  other  hand,  the  secondary  infection,  as.  e.  g.}  pneumonia,  erysipelas, 
etc.,  is  acquired  after  a  lapse  of  months  or  several  years,  or  if  the  second 
injection  of  serum  is  given  after  this  time,  i.  e.,  at  a  time  when  the  anti- 
bodies called  forth  by  the  primary  infection  or  first  injection  have 
disappeared,  a  certain  interval  of  time  will  elapse  before  symptoms  of 
sickness  develop,  as  in  the  case  of  the  first  group.  This  interval,  how- 


RELATION    OF   ANAPHYLAXIS   TO    INFECTIOUS   DISEASES  605 

ever,  instead  of  being  from  eight  to  twelve  days,  is  now  from  three  to 
seven,  a  fact  readily  explained  on  the  basis  that  a  cell  that  has  once  been 
stimulated  to  active  antibody  formation  will  subsequently  respond  to 
the  same  stimulus  with  increased  activity.  This  has  been  called  by 
von  Pirquet  the  accelerated  reaction. 

Group  III :  The  reaction  appears  immediately.  If  the  first  injection 
of  horse  serum  or  infection  is  followed  by  actual  disease  or  vaccination, 
the  reinjection  or  reinfection  is  acquired  at  a  time  when  the  antibodies 
are  present  in  the  circulation  in  considerable  amount,  a  reaction  will 
occur  either  immediately  or  within  the  first  twenty-four  hours.  This 
reaction  may  be  quite  virulent  in  intensity,  although  it  is  shorter  in 
duration  than  when  it  occurs  in  the  first  group,  von  Pirquet  speaks 
of  this  as  the  immediate  reaction.  It  is  to  be  observed  in  cases  of  serum 
sickness  where  the  symptoms  develop  almost  immediately  following  an 
injection  of  serum  months  and  even  years  after  a  previous  injection; 
it  also  occurs  in  cowpox  vaccination,  where  a  local  reaction  takes  place 
very  quickly  and  soon  disappears  after  a  previous  attack  of  smallpox 
or  vaccination. 

If  the  antibodies  are  present  in  lesser  amounts,  the  reaction  may  occur 
in  from  the  second  to  the  fourth  day;  this  is  called  the  torpid  early 
reaction. 

At  the  time  of  the  second  injection  of  serum  or  reinfection  with  bac- 
teria a  small  amount  of  antibody  may  still  be  present;  this  will  give  an 
immediate  though  mild  reaction,  and  is  not  enough  to  neutralize  the 
total  amount  of  foreign  protein  introduced.  A  portion  of  the  latter, 
therefore,  will  result  in  the  production  of  an  additional  amount  of  anti- 
body, which  occurs  in  an  accelerated  manner,  and  coming  in  contact 
with  some  of  the  free  antigen,  gives  rise  to  the  accelerated  reaction. 
Hence  we  may  have  an  immediate,  followed  by  an  accelerated,  reaction. 

To  illustrate  these  principles,  von  Pirquet  names  vaccinia  as  an 
example  of  an  acute  infection  in  which  the  processes  may  be  observed 
on  the  skin.  As  the  result  of  vaccination  a  colony  of  microorganisms 
is  formed  on  the  skin.  For  the  first  two  days  the  local  response  is 
evidently  traumatic  in  character.  After  the  third  or  fourth  day  the 
specific  reaction  sets  in,  i^i  the  form  of  a  small  papular  elevation  sur- 
rounded by  a  small  areola  due  to  the  local  action  of  toxins  or  protein 
poison  from  disintegrated  microorganisms.  By  the  eighth  day  a 
vesicle  has  formed,  and  from  its  contents  new  colonies  can  be  grown  on 
thousands  of  other  arms.  But  one  or  two  days  later  the  ferment-like 


606      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

antibody  appears.  The  colony  is  attacked,  its  contents  are  digested, 
a  toxic  substance  is  formed  that  diffuses  into  the  neighboring  tissues, 
and  the  intense  local  inflammation  which  we  call  the  areola  appears. 
In  addition  the  toxin  enters  the  general  circulation  and  fever  sets  in. 
Simultaneously  the  microorganisms  are  destroyed,  and  we  may  no 
longer  be  able  to  vaccinate  with  the  contents  of  the  now  yellow  pustule. 
After  two  or  three  days  the  real  struggle  is  ended,  although  the  local 
lesion  may  be  aggravated  by  secondary  infection,  and  the  body  contains 
the  new  antibody  for  a  long  time. 

If  we  now  revaccinate,  the  antibody  present  will  at  once  attack  and 
digest  the  microorganisms  introduced  into  the  scarification,  and,  as 
these  do  not  have  time  to  multiply,  only  an  extremely  small  amount  of 
toxin  is  formed,  which  gives  the  "immediate  or  early  reaction"  in 
vaccinia.  If  a  number  of  years  have  elapsed  between  the  first  and  the 
second  vaccination,  antibodies  may  be  absent  or  present  in  only  small 
amount,  but  the  body-cells  have  been  "keyed  up"  by  the  first  vac- 
cination, and  hence  react  more  quickly  to  the  second.  The  antibodies 
are  produced  in  from  three  to  five  days,  and  attack  the  microorganisms 
before  they  have  had  time  to  multiply  in  sufficient  numbers;  the  rel- 
atively small  amount  of  digestion  product  produces  a  comparatively 
mild  local  inflammation  and  practically  no  general  symptoms.  This  is 
sometimes  known  as  the  "immunity  reaction,"  or  vaccinoid,  and  is 
illustrated  in  Fig.  135. 

It  would,  of  course,  lead  us  too  far  were  we  to  analyze  all  the  different 
infectious  diseases  along  these  lines;  suffice  it  to  say  that  the  anaphy- 
lactic  principle  serves  to  explain  many  points  in  the  clinical  sympto- 
matology of  disease  that  were  not  explained  heretofore,  or  were  ascribed 
to  the  effects  of  the  bacteria  and  the  bacterial  products.  I  am  not 
among  those  who,  with  Vaughan,  would  ascribe  all  symptoms  to  the 
protein  poison  alone;  nor  do  I  regard  the  part  played  by  the  microorgan- 
ism as  simply  dependent  upon  whether  or  not  it  can  grow  in  the  body  and 
produce  the  ferment  antibody.  In  the  acute  toxemias,  for  instance,  such 
as  tetanus  and  diphtheria,  where  the  amount  and  toxicity  of  the  toxin 
are  out  of  all  proportion  to  the  number  of  bacteria,  and  where  the 
symptoms  develop  after  a  very  brief  incubation  period,  tissue  changes 
and  symptoms  are  in  all  probability  not  primarily  due  to  any  antigen- 
antibody  reaction,  with  liberation  of  the  protein  poison,  but  are  rather 
to  be  attributed  to  a  direct  action  of  the  toxin  upon  the  body-cells, 
especially  upon  those  for  which  the  toxin  possesses  a  special  affinity. 


RELATION    OF   ANAPHYLAXIS   TO    INFECTIOUS    DISEASES  607 

When  the  antibodies  appear,  we  may  rightfully  assume  that  a  ferment, 
in  the  nature  of  a  protein  amboceptor  or  lysin,  appears  with  the  antitoxin, 
and  theoretically  we  may  expect  a  clinical  reaction  due  to  the  protein 
poison  or  anaphylatoxin  liberated  from  the  bacilli.  It  may  be  that 
diphtheria  antitoxin  is  in  the  nature  of  this  ferment,  or  antitoxin  and 
ferment  may  exist  together  and  in  this  manner  afford  an  explanation 
for  the  local  destruction  of  the  bacilli  and  the  cleaning  up  of  the  mem- 
brane, the  effects  of  the  anaphylatoxin  being  overshadowed  by  the  true 
toxin,  or  expressing  itself  in  the  paralyses  and  other  symptoms  commonly 
ascribed  to  the  toxones.  von  Pirquet  concedes  that  in  scarlatina  the 
primary  symptoms  of  the  malady,  i.  e.,  the  eruption  and  angina,  are  due 
to  a  pure  toxin  effect, — to  the  true  scarlatinal  virus, — but  that  the  sequelae, 
and  notably  the  nephritis,  are  the  expression  of  the  action  of  anaphyla- 
toxins  that  are  formed  when  the  corresponding  antibodies  are  produced. 
Likewise  in  such  infections  as  typhoid  fever  and  pneumonia,  I 
cannot  eliminate  the  action  of  true  toxins  and  endotoxins,  and  ascribe 
all  the  symptoms  to  a  protein  poison  or  anaphylatoxin.  It  is  not  al- 
ways true  that  the  incubation  period  is  devoid  of  symptoms.  Mild 
and  evanescent  symptoms  are  frequently  present,  and  it  is  but  natural 
to  ascribe  these  to  the  effects  of  bacterial  products  themselves.  After 
a  time,  when  the  bacteria  have  multiplied  and  reached  special  tissues, 
the  symptoms  become  more  intense  and  the  typical  lesions  are  produced. 
These  may  be  ascribed  to  the  result  of  the  combined  action  of  toxins 
themselves  and  the  protein  poison,  rather  than  to  the  protein  poison 
itself  to  the  entire  exclusion  of  the  metabolic  products  of  the  bacteria. 
In  the  present  state  of  our  knowledge  it  is,  of  course,  very  difficult  to 
decide  which  symptoms  in  a  given  disease  are  due  to  bacterial  toxins, 
which  to  endotoxins,  which  to  ptomains,  which  to  a  mechanical  action 
of  the  bacteria,  and  which  to  the  protein  poison  or  anaphylatoxin. 
While  it  is  perfectly  true  that  we  can  now  understand  symptoms  as  due 
to  protein  poison  that  cannot  be  ascribed  entirely  to  toxins  and  endo- 
toxins, it  seems  to  me  unwise  to  ascribe  all  lesions  and  symptoms  to  the 
toxins,  on  one  hand,  or  the  protein  poison,  on  the  other.  It  is  necessary 
for  us  to  know  the  manner  in  which  the  protein  poison  is  produced, 
how  infection  is  so  closely  allied  to  what  we  know  as  anaphylaxis,  and 
that  in  any  one  disease,  such  as  diphtheria,  tetanus,  and  pneumonia,  the 
toxins  and  endotoxins  are  of  primary  and  immediate  importance,  whereas 
in  others,  such  as  smallpox,  chickenpox,  and  whooping-cough  the  protein 
poison  itself  is  of  primary  importance  and  that  in  all  disease  all  factors 
may  be  concerned  to  a  more  or  less  degree. 


608      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

RELATION  OF  ANAPHYLAXIS   TO  NON-INFECTIOUS  DISEASES 

When  we  come  to  consider  non-infectious  diseases, — and  by  this  I 
refer  particularly  to  the  symptom-complex  of  serum  disease  and  those 
conditions  commonly  ascribed  to  idiosyncrasies  toward  certain  sub- 
stances, such  as  horse  asthma,  satinwood  dermatitis,  buckwheat  poison- 
ing, urticarias  due  to  the  ingestion  of  strawberries,  pork,  and  the  like, — 
the  relation  of  anaphylaxis  to  the  processes  involved  is  more  intimate. 
Here,  indeed,  we  may  regard  the  symptoms  as  entirely  anaphylactic  in 
origin  and  character,  as  the  substances  in  themselves,  such  as  serum, 
egg-albumen,  horse  effluvia,  etc.,  are  not  regarded  as  toxic  and  we  have 
not  the  coincident  effects  of  toxins,  endotoxins,  and  ptomains,  as  in 
bacterial  infections. 

Here,  indeed,  we  may  more  readily  accept  Vaughan's  and  Fried- 
berger's  theory  as  to  the  action  of  the  protein  poison,  regarding  it  the 
same  in  all  proteins,  and  set  free  from  the  protein  molecule  by  the  so- 
called  "ferment,"  which  is  in  the  nature  of  a  protein  sensitizer  or  ambo- 
ceptor,  and  which,  with  a  complement,  brings  about  lysis  or  cleavage  of 
the  molecule,  just  as  a  similar  ferment  or  antibody,  called  bacteriolysin, 
disrupts  a  bacterial  cell  or  its  protein  constituents.  In  short,  we  may 
say  that  the  work  of  Vaughan  and  his  collaborators  has  shown  us  the 
presence  of  this  poison  in  all  protein  material  with  a  method  of  extracting 
it  in  vitro.  Friedberger  and  his  collaborators  have  shown  how  this 
poison. is  set  free  in  the  body  through  the  agency  of  the  "ferment." 
von  Pirquet  has  studied  the  question  at  the  bedside,  dispensing  with  the 
microscope  and  test-tube  and  depending  solely  upon  the  vital  processes 
and  reaction,  skilfully  combining  laboratory  findings  with  bedside  ob- 
servations— in  fact,  he  built  up  his  theory  on  the  nature  of  infection  and 
immunity  before  the  role  of  the  protein  poison  was  discovered,  and 
subsequent  work  has  added  support  to  his  conceptions.  Of  clinical  and 
practical  importance  in  this  connection  are  serum  disease  and  hyper- 
sensitiveness  or  idiosyncrasies  for  other  proteins  and  certain  drugs. 


SERUM  DISEASE 

This  name  was  applied  by  von  Pirquet  and  Schick1  to  the  various 
clinical  manifestations,  such  as  eruptions,  fever,  edema,  and  pain  in  the 
•joints,  following  the  injection  of  horse  serum.  These  symptoms  are  due 

1  "Die  Serumkrankheit,"  Leipsic,  Deuticke,  1905. 


SERUM    DISEASE  609 

to  the  horse  serum  itself,  for,  as  was  early  shown  by  Johannessan,1 
Bokay,2  and  others,  they  may  manifest  themselves  after  the  injection 
of  sterile  normal  horse  serum.  The  serum,  moreover,  of  certain  horses 
appears  to  be  more  likely  than  that  of  others  to  cause  these  symptoms, 
thus  accounting  for  the  fact  that  one  lot  of  antitoxin  will  cause  a  higher 
percentage  of  serum  sickness  than  will  another.  A  concentrated  serum 
is  not  so  likely  to  produce  serum  sickness  as  whole  serum,  owing  partly 
to  the  fact  that  smaller  doses  of  it  are  given.  According  to  Rolleston  and 
Ker,  the  frequency  of  serum  sickness  is,  as  a  rule,  in  direct  proportion  to 
the  amount  of  serum  given,  and  in  inverse  ratio  to  the  severity  of  the 
attack;  in  other  words,  we  may  expect  to  encounter  it  most  often  in 
mild  and  moderately  severe  cases  that  have  received  very  liberal  dosages 
of  serum. 

The  Nature  of  Serum  Disease. — Serum  sickness  is  regarded  as  a 
true  anaphy  lactic  phenomenon.  We  are  prone  to  call  the  severe,  fatal, 
and  rare  instances  of  death  following  serum  injection  examples  of  anaphy- 
laxis,  and  to  regard  serum  sickness  as  a  different  condition.  Both  are 
fundamentally  the  same,  except  that  in  the  first  instance  the  body-cells 
are  for  some  unknown  reason  unduly  and  highly  anaphy  lactic.  Fortu- 
nately, this  undue  hyper  sensitiveness  is  frequently  foreshadowed  by  the 
asthmatic  or  hay-fever-like  attacks  which  the  susceptible  person  may  exhibit 
when  he  enters  stables  or  is  otherwise  around  horses.  It  goes  without  saying 
that  horse  serum  should  never  be  given  to  such  persons. 

While  serum  sickness  is  usually  due  to  horse  serum  for  the  reason 
that  the  horse  is  so  commonly  employed  in  the  preparation  of  various 
curative  serums,  the  serum  of  the  ox,  rabbit,  and  other  animals  may  in- 
duce the  same  train  of  symptoms  in  addition,  in  some  instances,  to 
producing  a  direct  toxic  effect. 

An  immediate  reaction  rarely  follows  the  first  injection  of  serum  un- 
less the  patient  is  one  of  those  unfortunate  but  rare  persons  who  in  some 
manner  have  been  rendered  highly  sensitive  to  horse  protein.  In  the  ma- 
jority of  instances  symptoms  do  not  develop  for  from  eight  to  twelve 
days,  during  which  time  the  antibody  is  being  produced.  When  antibody 
formation  has  reached  a  certain  point,  it  reacts  upon  any  of  the  horse 
serum  that  may  persist  in  the  cells  or  circulation,  producing  the  anaphy- 
lactic  reaction.  If  the  dose  of  serum  has  been  small,  antibody  forma- 

1  Deutsch.  med.Wchnschr.,  1895,  21,  855. 

2  Jahresb.  f.  Kindesh.,  1897,  xliv,  133. 
39 


610      ANAPHYLAXIS    IN    RELATION   TO    INFECTION    AND    IMMUNITY 

tion  goes  on  as  usual,  but  the  serum  may  not  persist  in  the  circulation. 
Hence  symptoms  do  not  develop  in  such  a  person,  although  if  reinjected 
subsequently  symptoms  will  appear.  This  is  one  reason  why  a  con- 
centrated serum  is  not  so  likely  to  produce  serum  sickness,  since  a  smaller 
quantity  of  it  is  injected. 

If,  however,  the  patient  has  received  an  injection  of  serum  some 
months  previously,  a  reinjection  is  likely  to  be  followed  by  an  immediate 
reaction,  that  is,  the  symptoms  appear  within  from  twenty-four  to  forty- 
eight  hours.  If  the  first  injection  had  been  given  a  year  or  more  previ- 
ously, no  antibody  may  be  present  in  the  blood,  so  that  an  immediate 
reaction  does  not  occur.  If,  however,  the  cells  once  stimulated  are 
"keyed  up"  indefinitely,  and,  accordingly,  antibody  formation  is  quite 
rapid,  so  that  we  find  symptoms  developing  in  from  four  to  seven  days 
after  injection — the  accelerated  reaction.  Or  a  small  amount  of  antibody 
may  be  present  which  gives  a  mild  reaction  around  the  site  of  serum 
injection,  followed  in  from  four  to  seven  days  by  a  general  reaction, 
this  being  an  immediate  followed  by  the  accelerated  reaction. 

As  was  previously  stated,  anaphylaxis  is  specific — that  is,  a  person 
receiving  ox  serum  in  the  first  injection  would  not  be  affected  subse- 
quently by  an  injection  of  horse  serum,  but  only  by  ox  serum.  For  this 
reason  it  has  been  recommended  that  diphtheria  antitoxin  to  be  used  for 
prophylactic  purposes  should  be  prepared  by  immunizing  cattle,  re- 
serving the  horse  serum  for  treatment  if  the  disease  should  be  contracted 
subsequently. 

Symptoms  of  Serum  Disease. — The  most  typical  of  these  symptoms 
are  rash,  fever,  and  prostration,  besides  joint  and  muscle  pains,  edema, 
and  adenitis. 

The  most  obvious  and  important  of  the  symptoms  are  undoubtedly 
the  various  forms  of  rash.  In  1000  consecutive  cases  of  diphtheria 
treated  with  antitoxin  in  the  Philadelphia  Hospital  for  Contagious 
Disease,  a  rash  developed  in  430,  or  43  per  cent.  The  time  of  appear- 
ance of  the  eruption  depends,  as  was  just  stated,  upon  whether  or  not 
the  patient  has  been  injected  on  a  previous  occasion,  and  if  so,  the 
length  of  the  interval  between  the  first  and  subsequent  injection,  and 
to  a  lesser  extent  upon  the  amount  of  the  first  injection,  these  factors 
influencing  the  quantity  of  antibody  present  at  the  time  of  reinjection. 
Of  the  430  cases  just  mentioned,  the  time  of  appearance  of  the  rash,  in 
days,  after  subcutaneous  injection  of  antitoxin,  was  as  follows: 


x 


,  ;: 


FIG.  123. — URTICARIAL  RASH  OF  SERUM  SICKNESS. 

Case  of  laryngeal  diphtheria;  had  received  40,000  units  of  antitoxin  eight  days 
previously;  urticarial  rash  first  appeared  about  thirty  hours  before  this  drawing 
was  made. 


FIG.  124. — MULTIFORM  RASH  OF  SERUM  SICKNESS. 
Child  with  laryngeal  diphtheria  on  the  sixth  day  after  receiving  65,000  units  of 


antitoxin. 


SERUM   DISEASE 


611 


TABLE  22  —TIME  OF  APPEARANCE  OF  SERUM  RASH  IN  430  CASES  OF 

SERUM  DISEASE 


DAT  UPON  WHICH  THE  RASH  APPEARED  AFTER  INJECTION 
OF  DIPHTHERIA  ANTITOXIN 

TOTAL  NUMBER 
SHOWING  RASH 

PERCENTAGE 

First 

4 

0.9 

SBCOIK!                                               -        

6 

1.4 

Third                                               

7 

1.6 

Fourth                         

30 

6.9 

Fifth                    

35 

8.1 

Sixth                                                      • 

75 

17.6 

Seventh 

65 

15.1 

Eighth 

85 

19.8 

Ninth                               .            

44 

10.2 

Tenth                      

39 

9.0 

Eleventh               

22 

5.1 

Twelfth  

5 

1.1 

Thirteenth 

6 

1.4 

Fourteenth 

5 

1.1 

Fifteenth                                           .    . 

1 

0.2 

Sixteenth                  .              

1 

0.2 

In  this  table  are  included  some  cases  of  reinjection,  as,  e.  g.,  scarlet- 
fever  patients  who  received  a  routine  immunizing  dose  of  antitoxin 
upon  admission,  and  another  after  having  contracted  diphtheria;  also 
cases  of  diphtheria  that  became  reinfected  within  a  few  months  after 
their  discharge  from  the  hospital.  As  will  be  seen,  about  63  per  cent, 
of  cases  develop  a  rash  between  the  sixth  and  the  ninth  day  after  the 
injection  of  antitoxin. 

Three  main  types  of  rashes  are  generally  recognized : 

1.  Urticarial  Rashes. — If  we  include  in  this  group  all  eruptions  that 
present  a  resemblance  to  urticaria,  these  rashes  are  the  most  common, 
constituting  from  70  to  90  per  cent,  of  all  eruptions.     They  usually 
appear  after  the  seventh  day,  becoming  manifest  first  about  the  site  of 
injection.     Large,  irregularly  shaped,  and  scattered  blotches  appear, 
frequently  with  true  wheals  in  the  center  (Fig.  123),  accompanied  by 
intense  itching  and  irritation.     Sometimes  true  wheals  do  not  appear. 
The  rash  is  often  very  profuse,  and  fresh  blotches  may  continue  to  ap- 
pear for  two  or  three  days.     Occasionally,  the  rash  is  quite  sparse  and 
mild,  and  may  disappear  within  twenty-four  hours. 

2.  Multiform  Rashes. — This  type  of  rash  is  quite  common.     It  is 
often  circinate  in  its  arrangement,  or  occurs  in  large  blotches  mixed  with 
a  scattered  morbilliform  or  measly  form  of  rash  (Fig.  124).     Different 
parts  of  the  body  may  present  different  appearances  at  the  same  time. 
This  rash  may  occasionally  closely  simulate  true  measles,  especially 
since  it  involves  the  face,  and  as  the  conjunctive  are  likely  to  be  con- 
gested in  almost  any  variety  of  serum  sickness.     It  is  differentiated 


612      ANAPHYLAXIS   IN   RELATION   TO    INFECTION   AND    IMMUNITY 

from  measles  by  the  fact  that  Koplik's  spots  are  absent,  there  is  no  pro- 
dromal rise  in  temperature,  the  papules  are  not  elevated  above  the  skin 
as  much  as  in  measles,  and  that  the  eruption  frequently  starts  from  the 
site  of  injection,  instead  of  on  the  face. 

3.  Scarlatiniform  Rashes. — This  type  of  rash  occasionally  occurs, 
and  may  bear  so  close  a  resemblance  to  true  scarlet  fever  as  to  be  indis- 
tinguishable from  it.  The  eruption  usually  appears  early,  that  is,  in 
from  the  first  to  the  sixth  day  after  injection,  and  may  vary  from  a 
uniform  erythema  which  first  appears  about  the  site  of  injection,  to  a  true 
punctate  scarlatinal  rash.  The  differentiation  of  these  rashes  from  the 
true  scarlet-fever  eruption  is  one  of  the  greatest  sources  of  trouble  in 
hospital  practice,  especially  when  they  develop  in  the  diphtheria  wards, 
where  occasional  cases  of  true  scarlet  fever  are  always  likely  to  appear 
from  time  to  time.  The  absence  of  the  following  symptoms,  or  their 
occurrence  only  in  mild  degree,  would  favor  a  diagnosis  of  serum  disease : 
Fever,  or,  at  least,  the  presence  of  but  a  mild  pyrexia,  the  vomiting,  the 
typically  furred  tongue,  the  angina,  or  at  least  but  a  mild  throat  involve- 
ment, and  the  leukocytic  inclusion  bodies — all  forming  the  symptom- 
complex  of  true  scarlatina.  In  many  instances,  however,  it  is  necessary 
to  isolate  the  patient,  when  speedy  recovery,  absence  of  complications, 
and  less  definite  desquamation,  which  does  not  involve  the  palms  and 
soles,  indicate  that  the  patient  was  suffering  from  serum  disease  and  not 
from  scarlet  fever. 

Severe  Serum  Disease. — The  severer  forms  of  serum  disease, 
characterized  by  sudden  onset, — often  within  a  few  minutes  after  the 
injection  of  serum, — extreme  dyspnea,  prostration,  and  death  are, 
fortunately,  rare,  about  40  cases  in  all  being  now  on  record.  While 
physicians  are  justified  in  exercising  care  and  caution  in  the  administra- 
tion of  serum,  there  are  no  absolute  contraindications  to  its  use  except 
status  lymphaticus  and  those  instances  where  the  person  is  known  to  be 
hypersensitive  to  horse  serum.  Occasionally  sudden  and  severe  dyspnea 
and  prostration  accompany  an  immediate  reaction  when  a  reinjection 
of  serum  is  given  within  a  few  weeks  after  the  first.  Persons  suffering 
with  asthma  are  also  bad  risks,  especially  since  the  effects  of  serum  dis- 
ease upon  the  bronchial  mucosa  are  likely  to  aggravate  the  already  exist- 
ing condition.  This  subject  will  be  discussed  in  greater  detail  under  the 
head  of  Contraindications  to  Passive  Immunization  in  Chapter  XXX. 

The  Detection  and  Prevention  of  Serum  Disease. — Anaphylactic 
phenomena  can  usually  be  expected  to  occur  if  serum  is  given  within  a 
year  or  so  following  a  previous  injection.  Persons  who  experience  discom- 
fort when  about  horses  should  always  be  closely  questioned.  Figure  125 


FIG.  125. — LOCAL  SERUM  ANAPHYLACTIC  REACTIONS. 

Dr.  W.  P.,  anaphylactic  reactions  fifteen  minutes  after  application  of  serum; 
rabbit  serum  in  the  upper;  horse  serum  in  the  lower.  Subject  to  asthmatic  attacks 
when  in  an  animal  house  or  stable  where  rabbits,  horses,  and  guinea-pigs  are  kept. 


SERUM   DISEASE  613 

shows  the  urticaria-like  lesion  that  develops  on  the  arm  of  one  of  my  col- 
leagues following  scarification  and  the  thorough  application  of  a  drop  of 
horse  serum.  This  man  is  also  susceptible,  to  a  lesser  extent,. to  guinea- 
pig  and  rabbit  serum,  and  is  seized  with  sneezing  and  distressing  dyspnea 
after  .entering  a  house  where  these  animals  are  kept.  Thayer  has  re- 
ported a  case  of  buckwheat  hypersensitiveness  where  vaccination  with 
the  flour  resulted  in  a  local  reaction.  It  would  be  well  for  physicians 
to  make  this  simple  test  whenever  they  suspect  a  patient  of  being  hyper- 
sensitive to  horse  serum.  All  that  is  necessary  is  to  cleanse  the  arm  with 
alcohol,  scarify,  as  when  vaccinating  with  cowpox  virus,  and  rub  in  a 
drop  of  the  diphtheria  antitoxin  with  a  tooth-pick  or  some  suitable 
instrument.  The  reaction  usually  appears  within  fifteen  minutes,  and 
there  are  usually  no,  or  but  very  slight,  general  symptoms. 

Various  means  have  been  tried  and  advocated  to  bring  about  a  state 
of  anti-anaphylaxis  or  desensitization  of  the  patient : 

1.  A  preliminary  injection  of  0.5  c.c.  of  serum  as  antitoxin  may  be 
given,  followed  in  three  or  four  hours  by  the  regular  injection.    While 
it  is  difficult  to  produce  anti-anaphylaxis  (see  Chapter  XXIX),  this 
method  is  in  common  use. 

2.  According  to  Auer  and  Lewis,1  Auer,2  Anderson  and  Schultz,3 
Biedl  and  Kraus,4  and  Karsner,5  atropin  sulphate  has  a  distinct  protective 
action  against  the  asphyxia  of  acute  anaphylaxis  in  the  guinea-pig.     It 
may  be  well  to  administer  from  yiir  to  YQIT  grain  of  the  drug  hypoder- 
mically  before  injecting  the  serum,  and  once  or  twice  subsequently, 
at  intervals  of  twelve  hours,  in  those  cases  in  which  hypersensitiveness  is 
suspected,  especially  when  serum  has  been  injected  within  a  year's 
time. 

3.  Rectal  injections  of  serum  have  been  advised  by  Besredka,  who 
bases  his  recommendations  upon  the  fact  that  by  this  route  absorption 
takes  place  slowly  and  desensitization  is  gradual.     While  in  the  chapter 
on  Serum  Therapy  I  have  frequently  advised  the  intravenous  injection 
of  serum,  it  is  to  be  understood  that  this  applies  to  first  doses.     It  is 
far  more  dangerous  to  give  serum  intravenously  to  those  injected  on  a  former 
occasion;  in  these  instances  the  physician  will  do  well  to  give  his  injections 
intramuscularly  or  subcutaneously,  when,  if  symptoms  develop,  they  will 
not  be  explosive  nor  so  dangerous.     Since  rectal  injections  are  usually 
refused,  the  serum  may  be  administered  very  slowly  subcutaneously. 

1  Jour.  Amer.  Med.  Assoc.,  1909,  liii,  458;  Jour.  Exper.  Med.,  1910,  xii,  151. 

2  Amer.  Jour.  Physiol.,  1910,  xxvi,  439. 

3  Proc.  Soc.  Exper.  Biol.  and  Med.,  1910,  vii,  32. 

4  Wien.  klin.  Wochenschr.,  1910,  xxiii,  385. 
6  Jour.  Amer.  M.ed.  Assoc.,  1911,  Ivii,  1023. 


614      ANAPHYLAXIS   IN   RELATION   TO    INFECTION   AND   IMMUNITY 

Friedberger  and  Mita  have  advocated  the  slow  intravenous  infusion  of 
serum — a  drop  at  a  time. 

The  Treatment  of  Serum  Disease. — In  the  majority  of  instances  no 
treatment  is  necessary.  Calcium  chlorid  and  lactate,  in  doses  of  from 
3  to  5  grains,  have  been  advocated  as  a  prophylactic  and  as  a  cure,  but 
they  are  of  doubtful  utility.  Soothing  lotions,  a  brisk  cathartic,  and 
sedatives  are  indicated,  and  occasionally  an  opiate  is  advisable.  The 
administration  of  adrenalin  chlorid  by  subcutaneous  injections  frequently 
affords  quick  relief;  as  its  effects  are  of  short  duration,  repeated  doses 
may  be  necessary  until  the  urticarial  rash  has  subsided. 

In  the  rare  acute  attacks  marked  by  extreme  dyspnea  or  rapid  and 
shallow  breathing  with  rapid  and  feeble  pulse,  atropin  and  caffein  should 
be  administered  hypodermically. 


IDIOSYNCRASIES 

Studies  in  anaphylaxis  have  also  offered  an  explanation  of  many,  if 
not  of  all,  of  those  peculiar  instances  in  which  the  inhalation  of  some 
animal  effluvium  or  of  the  pollen  of  certain  plants  or  the  ingestion  of 
certain  food-stuffs  and  drugs  is  followed  by  a  train  of  symptoms,  among 
which  asthma  and  an  urticarial  rash  are  usually  quite  prominent. 
Hitherto  these  manifestations  have  not  been  understood,  and  were 
simply  classed  as  idiosyncrasies — a  term  that  is  correct  if  we  can  make  it 
mean  hyper  sensitiveness,  for  experimental  investigation  leaves  little 
doubt  but  that  in  these  persons  antibodies  for  the  substances  in  question 
are  present  in  the  body  cells  and  fluids  which  with  the  particular  protein 
when  it  gains  access  to  the  body  produces  the  anaphylactic  reaction 
responsible  for  the  symptoms.  One  remarkable  feature  of  these  instances 
of  idiosyncrasy,  however,  is  the  extreme  hypersensitiveness  of  the  body- 
cells,  especially  in  those  cases  where  the  inhalation  of  such  infinitesimal 
quantities  of  protein  as  are  contained  in  the  air  will  bring  on  a  typical 
asthmatic  attack  in  a  person  hypersensitive  to  horse  protein. 

Examples  of  idiosyncrasy  are  relatively  common.  Susceptible  per- 
sons learn  to  know  that  the  ingestion  of  this  or  that  substance  is  sure  to 
be  followed  by  various  distressing  symptoms.  How  and  when  these 
persons  became  hypersensitive  are  usually  not  known.  In  some  in- 
stances the  condition  is  found  in  one  or  both  parents  and  in  several 
members  of  the  same  family,  making  it  appear  to  be  hereditary.  It  is 
well  known  that  animals  may  be  sensitized  by  feeding  them  proteins 
not  usually  present  in  their  diet,  as  by  giving  guinea-pigs  horse  serum  or 
the  flesh  of  other  animals. 


IDIOSYNCRASIES  615 

Among  the  commoner  examples  of  this  form  of  allergy  or  anaphylaxis 
may  be  mentioned : 

1.  Horse  asthma,  observed  among  those  persons  who  are  seized  with 
sneezing,  cough,  dyspnea,  coryza,  and  prostration  when  they  come  near 
horses,  as  in  a  stable  or  when  driving. 

2.  Hay-fever,  first  ascribed  to  the  pollen  of  plants  by  Elliotson  in 
1831,  and  thoroughly  studied  by  Dunbar.     Wolff-Eisner  was  the  first 
to  regard  the  reaction  as  a  phenomenon  of  hypersensitivity  or  anaphy- 
laxis.    Individuals  subject  to  hay-fever  show  a  uniform  series  of  symp- 
toms at  certain  definite  seasons,  either  in  the  early  summer  or  in  autumn. 
These  are  a  reddening,  swelling,  and  watering  of  the  eyes,  sneezing,  a 
sore  feeling  in  the  throat  and  larynx,  and  asthmatic  disorders.     The 
instillation  into  the  eye  of  a  1  per  cent,  solution  of  pollen  in  physiologic 
salt  solution  is  usually  sufficient  to  elicit  a  typical  attack.     Certain 
persons  are  susceptible  to  various  weeds,  and  each  usually  knows  the 
particular  weed  to  avoid.     In  hay-fever  we  have  the  most  marked  in- 
stances of  extreme  hypersensitiveness;    persons  may  be  seized  with  an 
attack  when  some  distance  from  the  particular  weed  in  question.    Similar 

phenomena  are  observed  among  persons  susceptible  to  poison  ivy,  sumac, 
workers  in  satinwood  (satinwood  dermatitis),  and  other  forms  of  der- 
matitis venenata. 

3.  Food  Anaphylaxis.— Certain  foods,  as  eggs,  buckwheat  (phago- 
pyrismus),  pork,  oysters,  clams,  lobsters,  cheese,  strawberries,  goose- 
berries, and  even  vegetables,  may  act  as  poisons  when  ingested  by  per- 
sons who  are  hypersensitive  to  them.  The  symptoms  are  quite  varied 
and  are  not  all  understood,  as  the  subject  is  still  in  the  experimental 
stage  and  requires  much  investigation  from  both  the  laboratory  and 
clinical  sides.  Asthma  has  been  frequently  associated  with  a  condition 
of.  hypersensitiveness  or  allergy  to  certain  foods  by  Goodale,1  Smith,2 
Talbot,3  Schloss,4  and  others.  Symptoms  referable  to  the  alimentary 
tract  vary  from  a  feeling  of  "indigestion"  and  "heartburn"  to  severe 
diarrhea,  vomiting,  and  abdominal  pain.  Various  skin  diseases,  and  par- 
ticularly chronic  eczema,  urticaria,  and  angioneurotic  edema,  have  been 
ascribed  to  food  hypersensitiveness  by  Hazen,5  McBride  and  Shorer,6 
White,7  Strickler  and  Goldberg,8  and  Blackfan.9  The  withdrawal  of 

1  Boston  Med.  and  Surg.  Jour.,  1914,  clxx,  No.  22. 

2  Archiv.  Inter.  Med.,  1909,  3,  350. 

3  Boston  Med.  and  Surg.  Jour.,  1914,  171,  708. 

4  Amer.  Jour.  Dis.  Children,  1912,  3,  341. 
6  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  695. 

6  Jour.  Cutan.  Dis.,  1916,  34,  No.  2.  7  Jour.  Cutan.  Dis.,  1916,  34,  55. 

8  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvi,  249. 

9  Amer.  Jour.  Dis.  Children,  1916,  1,  441. 


616      ANAPHYLAXIS    IN    RELATION   TO    INFECTION    AND    IMMUNITY 

certain  food  or  foods  from  the  diet  of  hypersensitive  persons  has  resulted 
in  an  improvement  or  cure  of  an  eczema. 

The  diagnosis  of  food  idiosyncrasy  may  be  made  on  the  basis  of  the 
experience  of  the  patient  that  the  ingestion  of  a  certain  food  is  followed 
by  the  occurrence  of  certain  symptoms.  Cutaneous  and  intracutaneous 
skin  tests  have  also  been  employed,  and  apparently  with  success.  The 
cutaneous  tests  are  conducted  by  cleansing  the  skin  of  the  arm  with  alcohol 
and  abrading  a  small  area  of  skin  with  a  needle  or  von  Pirquet  borer. 
A  small  amount  of  the  food,  as  a  drop  of  egg-white  or  a  drop  of  a  5  per 
cent,  watery  solution  of  the  food,  is  now  applied  and  rubbed  into  the 
abraded  skin.  A  control  test  should  be  made  with  salt  solution  or  water 
alone,  as  persons  with  an  irritable  skin  may  react  with  slight  edema  and 
erythema  to  the  trauma.  A  positive  reaction  is  indicated  by  the  develop- 
ment of  a  well  defined  urticarial  wheal  surrounded  by  a  zone  of  erythema 
and  similar  to  the  cutaneous  serum  reaction  shown  in  Fig.  123.  The 
reaction  appears  within  five  to  ten  minutes  and  lasts  from  twenty  to 
forty-five  minutes. 

The  intracutaneous  test  is  more  delicate  but  somewhat  more  painful 
and  likely  to  yield  confusing  and  non-specific  reactions.  It  consists 
in  the  intracutaneous  injection  of  a  very  minute  amount  of  a  sterile 
solution  of  the  protein  of  the  food;  usually  0.1  c.c.  of  0.1  per  cent,  solu- 
tion suffices.  The  reactions  should  not  be  read  until  after  the  elapse  of 
forty-eight  hours,  as  non-specific  reactions  may  be  observed  during  the 
first  twenty-four  hours  after  injection.  Positive  reactions  are  indicated  by 
the  formation  of  a  tender  papule  with  well-marked  edema  and  erythema. 

As  a  general  rule  a  series  of  tests  with  different  foods  may  be  done 
with  the  skin  of  both  arms;  soluble  proteins  of  various  foods  are  marketed 
in  a  convenient  form  for  the  tests. 

The  treatment  of  food  idiosyncrasy  consists  in  the  withdrawal  of  the 
food  from  the  diet  and  attempts  toward  desensitization  by  feeding  the 
food  in  very  small  and  non-toxic  doses  until  the  skin  reaction  disappears 
or  becomes  very  weak.  If  a  person  is  very  sensitive  it  is  well  to  begin . 
with  a  dose  of  less  than  0.01  gram  three  times  per  day,  gradually  in- 
creasing until  the  food  may  be  ingested  without  apparent  harm.  The 
process  of  desensitization,  however,  is  prolonged,  and  in  my  experience 
has  not  been  accomplished  in  several  cases  of  extreme  hypersensitiveness 
to  egg-albumen.  Attempts  have  also  been  made  to  desensitize  by  sub- 
cutaneous injections  of  a  sterile  solution  of  the  particular  protein  con- 
cerned. The  initial  doses  should  be  small  and  approximately  the  smallest 
amount  capable  of  eliciting  an  intracutaneous  reaction. 

4.  Certain  drugs,  such  as  iodoform,  iron  citrate,  and  even  atropin, 


IDIOSYNCRASIES  617 

strychnin,  morphin,  etc.,  appear  to  develop  a  state  of  hypersensitive- 
ness.  My  colleague,  Dr.  Fred  Boerner,  who  is  hypersusceptible  to 
quinin  and  who  suffers  acutely  following  the  swallowing  of  small 
amounts,  has  recently  shown  a  reaction  in  his  skin  following  the  appli- 
cation of  quinin  to  an  abrasion.1  Klausner  was  able  passively  to  sensitize 
guinea-pigs  against  iodoform  by  injecting  the  serum  of  a  person  sensitive 
to  this  drug.  Examples  of  drug  anaphylaxis  or  idiosyncrasy  are  more 
difficult  to  explain  unless  such  drugs  contain  a  protein  substance.  On 
the  other  hand,  a  drug  may  alter  a  body  protein,  rendering  it  really  a 
foreign  protein,  and  this  may  sensitize  body-cells  in  the  same  manner 
as  in  the  "indirect  anaphylaxis"  of  Richet,  referred  to  in  the  preceding 
chapter,  following  the  second  chloroforming  of  a  dog. 

As  was  previously  stated,  anaphylaxis,  or  rather  the  anaphylactic 
mechanism  on  the  basis  of  the  humoral  theory,  may  be  considered  one  of 
the  essential  steps  in  affording  resistance  to  disease  or  the  state  of  im- 
munity. Broadly  speaking,  the  lesions  and  symptoms  of  infection  may  be 
ascribed  to  the  effects  of  soluble  toxins,  endotoxins,  and  a  protein  poison. 
According  to  our  present  knowledge  the  endotoxins  are  mainly  liberated 
with  lysis  or  disrupture  of  the  bacterial  cell.  Similarly,  the  protein  poison 
is  produced  by  cleavage  of  the  bacterial  protein  substance.  Whether  the 
endotoxins  and  protein  poison  are  identical  it  is  impossible  to  state. 
For  the  present  it  may  be  well  to  consider  them  as  separate  entities. 
In  certain  infections,  such  as  tetanus  and  diphtheria,  the  soluble  toxins 
are  chiefly  concerned,  and  these  are  neutralized  by  specific  antibodies, 
the  antitoxins.  The  antitoxins  are  not  similar  to  the  " ferment"  that 
splits  protein,  because  they  are  able  to  neutralize  their  toxins  without 
the  aid  of  complement.  In  other  infections,  such  as  typhoid  fever, 
cholera,  and  pneumonia,  the  endotoxins  and  protein  poison  may  be  con- 
sidered the  main  etiologic  factors.  The  chief  antibodies  are  a  cytolysin 
(bacteriolysin),  which  disrupts  or  kills  the  bacterial  cells,  and  bacterio- 
tropin,  which  brings  about  the  same  result  by  favoring  phagocytosis. 
Apparently  the  cytolysins  and  the  so-called  " ferments"  responsible  for 
cleaving  the  protein  substance  are  quite  similar  in  their  mechanism. 
The  former  are  amboceptors,  thermostabile  and  inactive  without  the 
presence  of  a  complement.  There  is  no  doubt  but  that  heating  a  serum 
containing  a  bacteriolysin  and  a  complement  will  render  the  serum  in- 
active through  destruction  of  the  complement.  While  the  ferment  con- 
cerned in  splitting  protein  is  regarded  by  many  as  an  amboceptor  and 
complement,  there  is  no  general  agreement  on  this  point.  Some  in- 
vestigators, for  example,  believe  that  the  ferment  concerned  in  splitting 
1  Jour.  Amer.  Med.  Assoc.,  1917,  Ixviii,  907. 


618      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

placental  protein,  as  in  Abderhalden's  pregnancy  reaction,  is  rendered 
totally  inactive  by  heating  the  serum.  Others  ^believe  that  heating 
diminishes  the  activity  of  the  ferment,  but  does  not  destroy  it  altogether; 
the  point  cannot  be  decided  at  present  mainly  because  of  technical 
difficulties,  but,  for  the  sake  of  simplicity  at  least,  we  may  regard  the 
protein-splitting  ferments  as  quite  similar  to  the  cytolysins;  indeed, 
they  may  be  identical. 

In  anaphylaxis  we  recognize  two  primary  factors:  first,  the  anti- 
body, which  acts  with  the  protein  substance,  which  may  be  either  a  harm- 
less sterile  protein,  such  as  horse  serum  or  pathogenic  bacteria,  with  the 
production  of  anaphylaxis;  second,  a  state  of  hypersensitiveness  of  the 
body-cells.  While  it  is  clearly  apparent  that  the  protein  poison  generated 
in  the  test-tube  may  intoxicate  normal  animals  with  the  first  injection, 
yet  to  understand  the  extreme  sensitiveness  of  body-cells  in  persons 
susceptible  to  horse  protein,  where,  for  example,  a  few  inspirations  of 
stable  air  are  sufficient  to  bring  on  an  attack  of  asthma,  we  must  recog- 
nize a  peculiar  hypersensitiveness  of  these  cells,  due  probably  to  the 
fact  that  protein  amboceptors  are  attached  to  the  cells  and  unite  with 
the  inhaled  protein  with  great  avidity. 

The  relation  of  anaphylaxis  to  immunity  consists,  therefore,  in  the 
fact  that  the  mechanism  concerned  in  anaphylaxis  is  apparently  related 
with  that  concerned  in  antibacterial  immunity.  Vaughan  believes  that 
the  same  mechanism  is  identical  in  all  forms  of  immunity,  but  we  can- 
not subscribe  to  this  view  because  the  mechanism  concerned  in  anaphy- 
laxis does  not  explain  antitoxin  immunity,  or  at  least  the  antibodies 
concerned  in  neutralizing  diphtheria  toxin  are  different  from  those 
digesting  or  splitting  a  bacterial  protein,  as,  for  example,  typhoid  bacilli. 
In  antibacterial  immunity,  however,  where  the  chief  action  lies  in  digest- 
ing the  infecting  cells,  the  mechanism  may  be  regarded  as  similar  with 
that  concerned  in  producing  the  anaphylatoxin  or  protein  poison  of  the 
humoral  theory.  The  effects  are,  however,  different.  In  infection  we 
have  the  combined  action  of  toxins,  endotoxins,  and  protein  poison  upon 
the  body-cells;  in  serum  anaphylaxis  we  have  the  effects  of  the  protein 
poison  alone.  Lesions  and  symptoms  of  disease,  therefore,  may  be 
regarded  as  the  summation  of  the  products  of  infection  and  anaphylaxis. 

When  we  inject  a  bacterial  vaccine  we  inject  so  much  bacterial  pro- 
tein. This  protein  sensitizes  body-cells  and  causes  them  to  produce  an 
amboceptor  (sensitizer  or  the  anaphylactic  ferment);  this  antibody 
serves  to  bring  about  death  by  lysis  of  any  corresponding  bacteria  in  the 
body  (therapeutic  immunization),  or  of  any  that  may  subsequently 
gain  access  (prophylactic  immunization).  The  effects,  if  apparent, 


ANAPHYLACTIC    OR   ALLERGIC    REACTIONS  619 

may  be  ascribed  to  the  protein  poison  liberated,  also  to  the  liberated 
endotoxins,  if  we  consider  these  as  separate  from  the  protein  poison. 

In  antibacterial  immunity,  therefore,  we  recognize  lysis  or  digestion 
of  the  infecting  bacteria  by  an  antibody  as  the  chief  means  of  defense. 
This  antibody  is  produced  by  previous  injection  of  the  bacterial  protein 
in  the  form  of  a  vaccine,  or  as  the  result  of  a  previous  infection.  This 
is  true  also  of  serum  anaphylaxis,  or  of  egg,  milk,  pollen,  or  any  other 
form  of  anaphylaxis,  and  in  this  way  anaphylaxis  is  brought  into  rela- 
tion with  immunity. 

With  the  antibody  in  our  body-fluids,  the^  corresponding  bacterium 
is  destroyed  soon  after  it  comes  into  contact  with  the  antibody,  but  since 
the  amounts  of  protein  poison  and  endotoxin  released  are  small  and 
highly  diluted,  we  experience  none  or  but  slight  effects.  If,  however, 
the  infection  or  entrance  of  bacterial  protein  is  strictly  localized  to  a 
small  area,  so  that  the  liberated  poisons  are  concentrated,  a  local  reac- 
tion is  produced,  such  as  is  seen,  for  example,  in  the  cutaneous  tubercu- 
lin, luetin,  mallein,  and  similar  reactions. 

The  question  may  now  arise  as  to  the  manner  in  which  the  body 
cells  dispose  of,  or  become  accustomed  to,  or  are  protected  from  the 
protein  poison  and  endotoxin.  There  is  no  satisfactory  answer  to  this 
question.  We  have  seen  that  repeated  injections  of  the  same  protein 
lead  to  a  condition  of  decreased  sensitiveness.  The  method  by  which 
endotoxins  and  the  protein  poison  are  neutralized,  and  whether  anti- 
bodies for  these  are  produced,  are  points  that  require  further  investiga- 
tion. This  subject  has  been  discussed  in  the  preceding  chapter,  under 
the  head  of  Anti-anaphylaxis.  It  is  true  that  our  body-fluids  may  con- 
tain large  amounts  of  the  antibody;  that  protein,  bacterial  or  other, 
may  be  vigorously  split,  but  still  we  do  not  suffer  from  the  effects  of  the 
protein  poison  or  endotoxins.  Whatever  the  mechanism,  it  in  some 
manner  concerns  a  neutralization  or  depression  of  the  susceptibility  or 
hypersensitiveness  of  the  body-cells;  either  the  protein  antigen  is  stored 
in  the  cells  and  in  some  manner  depresses  cellular  activity,  as  Weil 
suggested,  or  else  the  protein  is  split  beyond  the  toxic  moiety  by  the 
free  amboceptor  in  the  body-fluids,  and  in  this  manner  prevents  the 
protein  poison  from  reaching  the  sessile  receptors  (those  amboceptors 
still  attached  to  the  body-cells).  This  reasoning  is  based  upon  the 
assumption  that  symptoms  are  produced  only  in  case  the  poison  be- 
comes attached  to  the  cells,  according  to  the  cellular  theory  of  anaphy- 
laxis (see  the  preceding  chapter). 


620      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

ANAPHYLACTIC  OR  ALLERGIC  SKIN  REACTIONS 

As  was  previously  stated,  if  a  protein,  such  as  tubercle  potein 
(tuberculin),  syphilis  protein  (luetin),  glanders  protein  (mallein),  etc., 
is  concentrated  and  applied  to  or  injected  into  the  skin  or  mucous  mem- 
brane in  a  local  sera,  and  if  the  body-cells  have  been  rendered  anaphy- 
lactic to  this  protein,  a  local  reaction  is  produced  characterized  chiefly 
by  congestion  and  edema.  This  local  reaction  is  regarded  as  analogous 
to  the  urticarial  or  other  eruptions  accompanying  general  serum  anaphy- 
laxis  (serum  disease)  and  is  regarded  as  an  anaphylactic  or  allergic 
reaction,  although  there  aje  not  sufficient  data  at  hand  to  prove  that  the 
mechanism  of  the  local  or  skin  reaction  is  identical  with  that  of  the 
general  and  severe  reaction  which  may  follow  an  intravenous  injection 
of  the  protein. 

Etiology  of  Skin  Reactions. — Skin  reactions  are  conducted  by  intra- 
dermal  injection;  by  application  to  an  abrasion -of  the  skin;  by  rubbing 
into  the  intact  skin  or,  as  on  mucous  membranes,  by  mere  contact,  as 
instillation  into  the  conjunctival  culdesac.  In  the  first  and  second 
methods  trauma,  due  to  the  operation  itself,  is  a  factor  in  the  production 
of  the  resulting  inflammation;  likewise  the  intracutaneous  injection  of 
practically  any  protein  or  non-protein  substance  will  elicit  a  non-specific 
inflammatory  reaction  providing  the  dose  injected  is  large  enough.  I  have 
summarized  the  probable  causes  of  various  skin  reactions  as  follows1: 

1.  The  true  anaphylactic  skin  reaction,  a  specific  process  due  to  the 
interaction  of  specific  anaphylactic  antibody  and  specific  anaphylac- 
togen,  largely  within  or  upon  the  cells  and  with  the  formation  of  a  diffus- 
ible irritant  or  the  production  of  cellular  shock,  capable  of  producing 
acute  hyperemia,  edema,  and  leukocytic  infiltration  of  the  skin. 

2.  The  traumatic  and  non-specific  protein  skin  reactions  which  may 
be  caused  (a)  by  trauma  and  the  direct  injection  of  an  irritant  used  as  a 
preservative  for  the  material,  as  phenol,  or,  to  a  preformed  toxic  and 
irritant  substance,  as  diphtheria  toxin  in  the  Schick  test;   (6)  to  the 
production  of  a  protein  poison  of  irritant  qualities  b}*-  the  action  of 
non-specific  proteolytic  ferments  in  the  serum  or  derived  from  injured 
cells,  upon  the  protein  of  the  patient's  serum,  devitalized  cells,  injected 
protein,  or  all  three. 

The  intradermal  test  has  proved  the  most  delicate  means  of  eliciting 
a  local  anaphylactic  response,  but  is  also  more  likely  to  yield  the  non- 
specific and  traumatic  reactions.  Physicians  conducting  skin  reactions 
should  carefully  bear  in  mind  the  possibility  of  mistaking  a  pseudo-  or 
traumatic  reaction  for  the  true  anaphylactic  reaction,  and  also  that 
1  Bull  Johns  Hopkins  Hosp.,  1917,  xxviii,  163. 


ANAPHYLACTIC    OR   ALLERGIC    SKIN    REACTIONS  621 

certain  drugs,  particularly  the  iodids  and  bromids,  may  so  favor  the 
non-specific  reaction  as  to  present  evidences  of  a  violent  reaction  in 
normal  persons  which  may  be  interpreted  as  anaphylactic  (Sherrick,1 
Kolmer,  Matsunami  and  Broadwell,2  Kolmer,  Matsunami,  Immerman 
and  Montgomery3). 

Anaphylactic  Skin  Reactions  as  Indices  of  Infection  and  Hyper- 
susceptibility. — As  is  well  known,  anaphylactic  skin  reactions  may  be 
elicited  in  various  bacterial  and  protozoan  diseases  with  anaphylactogens 
prepared  from  the  protein  of  the  respective  microparasites,  particularly 
in  tuberculosis,  glanders,  typhoid  fever,  and  syphilis.  Well-marked  and 
specific  reactions  have  also  been  found  by  Amberg4  and  Kolmer  and 
Strickler  in  ringworm  and  favus,  and  isolated  reports  show  that  they 
may  be  elicited  in  various  other  diseases  with  the  proper  preparations. 

In  these  conditions,  however,  the  skin  reactions  are  not  always 
elicited,  and  especially  during  the  early  and  acute  stages.  An  interval 
of  time  is  required  for  the  purpose  of  sensitization,  and  during  the  acute 
stages  antigen  and  antibody  may  both  be  present  in  the  cells  and  body 
fluids,  as  shown  by  Weil5  and  Denzer,6  with  continual  interaction  ex- 
pressed in  the  symptom-complex  of  the  infection,  and  thereby  giving  no 
response  when  the  protein  is  applied  or  injected  into  the  skin.  Further- 
more, the  chemical  nature  of  the  protein  of  our  anaphylactogen  may  ha,ve 
been  altered  in  the  course  of  preparation  to  a  sufficient  extent  to  fail 
to  elicit  an  anaphylactic  reaction.  These  and  other  factors  not  under- 
stood diminish  the  practical  value  of  a  skin  test.  At  present,  however,  it 
may  be  stated  that  they  possess  a  diagnostic  value  which  is  particularly 
high  in  chronic  infections,  and  that  no  other  satisfactory  clinical  or 
laboratory  test  for  the  state  of  anaphylaxis  to  a  perticular  protein 
and  for  the  anaphylactic  antibody  has  been  discovered  except  that  by 
which  the  protein  is  actually  brought  into  relation  with  the  body  cells, 
as  in  the  parenteral  introduction  of  the  protein.  Of  great  interest  in 
this  connection  is  the  relation  of  the  intensity  of  the  anaphylactic  skin 
reaction  to  the  extent  of  the  infection.  Krause7  has  recently  studied 
in  a  thorough  and  excellent  manner  the  tuberculin  skin  reaction  in 
relation  to  experimental  tuberculosis,  finding  that  cutaneous  hyper- 
sensitiveness  to  tubercle  protein  is  inaugurated  by  the  establishment 
of  infection  and  the  development  of  the  initial  focus;  that  the  skin 

1  Jour.  Amer.  Med.  Assoc.,  1915,  July  31,  404. 

2  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  718. 

3  Jour.  Lab.  and  Clin.  Med.,  1917,  ii,  401.          4  Jour.  Exper.  Med.,  1910,  xii,  435. 

5  Proc.  Soc.  Exper.  Med.  and  Biol.,  1914,  xii,  37. 

6  Jour.  Infect.  Dis.,  1916,  xviii,  631. 

7  Jour.  Med.  Research,  1911,  xxiv,  361;  ibid.,  1916,  35,  1;  25. 


622      ANAPHYLAXIS    IN    RELATION   TO    INFECTION    AND    IMMUNITY 

reaction  increases  with  progressive  disease;  is  diminished  with  healing 
and  increased  by  reinfection. 

The  clinical  significance  and  practical  value  of  skin  reactions  are 
largely  of  a  diagnostic  nature  for  the  detection  of  hypersensitiveness 
to  a  protein  or  proteins,  which  may,  when  introduced  into  the  organism, 
produce  various  acute  or  chronic  lesions  and  symptoms  of  disease. 

Anaphylactic  Skin  Reactions  as  Indices  of  Immunity. — Of  further 
importance  is  the  question  of  the  clinical  significance  of  a  local  anaphy- 
lactic  reaction  as  an  index  of  immunity,  that  is,  resistance  to  an  infection 
or  reinfection.  Is  the  anaphylactic  antibody  capable  of  attacking 
and  destroying  the  antigenic  protein  in  a  living  state?  Are  protective 
and  curative  antibodies  produced  by  the  defensive  mechanism  of  the 
body  while  the  cells  are  being  sensitized  and  the  anaphylactic  antibody 
is  being  produced?  In  other  words,  is  hypersensitiveness  to  be  regarded 
as  an  index  of  resistance? 

Romer1  and  Sata,2  in  experiments  among  cattle  with  Bacillus  tuber- 
culosis, reached  the  conclusion  that  a  state  of  hypersensitiveness  meant 
a  certain  degree  of  resistance,  while  Krause3  and  Austrian4  have  ex- 
pressed the  opinion,  based  upon  experiments,  that  sensitization  of  non- 
tuberculous  animals  with  tubercle  protein  does  not  raise  their  resistance 
to  experimental  tuberculosis  infection,  and,  indeed,  may  lower  it. 

More  recently  Gay  and  Force5  have  greatly  renewed  interest  in  this 
subject  by  advocating  the  skin  test  as  a  means  of  determining  defensive 
activity  following  typhoid  fever  or  active  immunization  by  means  of 
vaccines.  Their  first  work  was  conducted  with  a  "typhoidin"  prepared 
in  the  same  manner  as  Koch's  old  tuberculin  by  cutaneous  inoculation. 
Later  Gay  and  Claypole6  prepared  typhoidin  by  precipitating  the  so- 
lution with  alcohol,  washing  the  precipitate  with  alcohol  and  ether, 
drying  in  a  vacuum,  and  suspending  the  resulting  powder  in  phenolized 
normal  salt  solution,  which  was  injected  intracutaneously  and  applied 
cutaneously,  a  control  powder  being  prepared  from  broth  and  used  in 
the  same  manner.  With  this  skin  test  Gay  and  his  associates  have 
studied  the  relative  value  of  various  vaccines  and  regard  the  anaphy- 
lactic reaction  as  indicative  of  a  state  of  immunity.  Nichols7  has 
questioned  the  value  of  the  anaphylactic  skin  test  as  an  index  of  im- 
munity and  regards  the  typhoidin  reaction  as  indicating  nothing  more 

1  Beitr.  z.  Klinik.  d.  Tuberk.,  1908,  xi,  79.  2  Ztsch.  f.  Tuberk.,  1911,  xviii,  1. 

3  Jour.  Med.  Research,  1911,  xxiv,  361;  ibid.,  1916,  35,  1;  35. 

4  Bull.  Johns  Hopkins  Hosp.,  1913,  xxiv,  11. 

5  Archiv.  Int.  Med.,  1914,  xiii,  471.  6  Archiv.  Inter.  Med.,  1914,  xiv,  671. 
7  Jour.  Exper.  Med,.  1915..  xxii,  780. 


TUBERCULIN    REACTION  623 

than  sensitization  to  typhoid  protein,  which  is  apparently  less  lasting 
and  less  specific  than  the  true  immunity  to  this  infection. 

The  experiments  of  my  associates  and  I1  in  this  field  have  been 
largely  tests  in  vitro  for  various  antibodies,  as  agglutinins,  bacteriolysins, 
and  complement-fixing  substances  in  the  fresh  sterile  blood  sera  of 
persons  and  lower  animals  hypersensitive  to  various  proteins  (typhoid, 
syphilis,  diphtheria,  and  canine  distemper),  and  have  shown  that  the 
state  of  hypersensitiveness  to  a  particular  bacterial  protein  bears  no 
relation  to  the  presence  or  absence  of  demonstrable  amounts  of  these 
antibodies.  The  experiments  of  Meyer2  have  corroborated  our  results 
and  conclusions,  and  have  furthermore  shown  that  a  positive  typhoidin 
skin  reaction  in  a  rabbit  does  not  indicate  the  presence  of  resistance 
to  an  infection  with  Bacillus  typhosus. 

The  sum  total  of  these  studies  indicate  that  although  antibodies 
that  may  be  regarded  as  possessing  protective  and  curative  properties 
toward  a  certain  protein  may  be  present  in  the  body  fluids  of  persons 
and  animals  hypersensitive  to  this  particular  protein,  the  condition  of 
hypersensitiveness  in  itself  is  no  direct  evidence  of  their  presence  of 
resistance  to  a  particular  infection,  although  these  antibodies  are  most 
likely  to  be  present  in  the  body  fluids  of  those  persons  who  are  hyper- 
sensitive. The  positive  anaphylactic  skin  test  is,  therefore,  evidence 
of  infection  or  sensitization  to  a  particular  protein  probably  without 
bearing  any  direct  relation  to  resistance  to  infection  or  reinfection. 

TUBERCULIN  REACTION 

An  account  of  Koch's  discovery  of  tuberculin,  in  1891,  is  given  in 
the  chapter  on  Tuberculin  Therapy.  Suffice  it  to  say  here  that  Koch 
was  most  interested  in  the  curative  properties  of  tuberculin,  and  while 
he  has  accurately  and  clearly  described  the  classic  picture  of  the  syste- 
matic tuberculin  reaction,he  failed  to  apprec  iate  the  true  significance  of  the 
reaction  at  the  site  of  injection,  although  its  occurrence  is  carefully  noted. 

The  Tuberculin  Reaction. — The  reaction  to  tuberculin  is  character- 
ized by  three  essential  features: 

1.  A  constitutional  reaction,  consisting  of  fever  and  the  accompany- 
ing general  symptoms  of  lassitude,  anorexia,  and  rapid  pulse,  varying 
in  severity  with  the  intensity  of  the  reaction. 

2.  A  local  reaction  at  the  site  of  administration,  varying  in  intensity 
from  slight  tenderness  and  redness  to  severe  inflammation  with  adenitis. 

3.  A  focal  reaction  about  the  tuberculous  lesion. 

1  Jour.  Immunology,  1916,  1,  409;  ibid.,  1916,  1,  429;  ibid.,  1916,  1,  443;  ibid., 
1916,  1,  571. 

2  Jour.  Infect.  Dis.,  1917,  xx,  424. 


624      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

These  reactions  do  not  by  any  means  run  parallel.  An  intense 
local  reaction  may  occur,  with  no  or  but  slight  constitutional  disturbance. 
Not  infrequently,  and  particularly  in  slight  pulmonary  lesions,  signs 
indicating  a  focal  reaction  may  not  be  elicited. 

Nature  of  the  Tuberculin  Reaction. — Koch  believed  that  the  tu- 
berculin reaction  was  due  to  a  summation  of  the  effects  of  the  injected 
toxin  and  the  toxic  bodies  formed  by  the  tubercle  bacillus  within  the 
infected  host.  Koehler  and  Westphal,  in  1891,  suggested  that,  by  a 
union  of  the  tuberculin  with  the  products  of  the  tubercle  bacillus,  a 
third  new  body  was  formed  in  the  tuberculous  focus.  Marmorek,  in 
1894,  suggested  that  the  tuberculin  stimulated  the  tubercle  bacilli  to 
secrete  a  fever-producing  substance. 

Finally,  in  1903,  von  Pirquet  and  Shick  explained  the  reaction  as 
due  to  a  "  vital  antibody  reaction,"  and  this  explanation  is  the  one  most 
generally  accepted  to-day.  According  to  this  conception,  an  antibody- 
like  substance  produced  by  the  bacilli  and  diffused  through  the  tissues 
enters  into  combination  with  the  tuberculin,  giving  rise  to  the  formation 
of  a  toxic  substance  in  the  general  circulation,  as  well  as  at  the  point  of 
inoculation  of  the  tuberculin,  von  Pirquet's  discovery  of  the  cutaneous 
reaction,  in  1907,  was  a  result  of  this  theory,  and  served  to  establish  a 
further  analogy  between  cowpox  vaccination,  tuberculosis,  and  the 
tuberculin  reaction.  According  to  the  principles  laid  down  in  the  earlier 
portion  of  this  chapter,  the  tubercle  bacilli  are  considered  as  stimulat- 
ing the  body-cells  to  produce  an  antibody  or  a  " ferment"  in  the  nature 
of  an  amboceptor,  which  splits  the  tubercle  protein  contained  in  tu- 
berculin, liberating  a  protein  poison,  which  produces  a  general,  local, 
and  focal  reaction.  In  terms  of  the  cellular  theory  of  anaphylaxis  the 
effects  are  due  to  the  interaction  of  tuberculin  and  antibodies  in  the 
cells  (Weil1). 

The  general  reaction  may  be  explained  as  due  to  a  general  effect  of 
the  poison  on  body-cells.  The  local  reaction  is  caused  by  a  concentra- 
tion of  the  poison  at  the  site  of  administration  of  the  tuberculin,  and  the 
focal  reaction  is  due  to  the  fact  that  cells  about  the  lesions  are  more 
sensitive  to  the  effects  of  the  poison  than  are  other  cells,  probably  because 
they  are  most  concerned  in  antibody  production  and  are  supplied  with  a 
large  number  of  sessile  or  attached  receptors  (amboceptors)  for  the 
tuberculin. 

Specificity  of  the  Tuberculin  Reaction. — The  tuberculin  reaction  is 
highly  specific.  This  does  not  mean  that  every  case  of  tuberculosis 
will  give  a  tuberculin  reaction,  and  positive  reactions  are  occasionally 
1  Jour.  Amer.  Med.  Assoc.,  1917,  Ixviii,  972. 


TUBERCULIN   REACTION  625 

found  in  apparently  healthy  persons  and  cattle.  The  conditions  under 
which  a  negative  reaction  may  occur  in  the  presence  of  tuberculosis 
are,  however,  fairly  well  understood,  and  physicians  should  be  thoroughly 
acquainted  with  these.  Likewise  most  instances  in  which  a  positive 
reaction  was  observed  in  the  apparent  absence  of  tuberculosis  have 
usually  narrowed  down  to  the  fact  that  the  lesion  was  so  small  or  so 
situated  as  to  escape  detection,  and,  indeed,  this  has  been  shown  so 
conclusively  by  autopsies  that,  in  the  presence  of  a  tuberculin  reaction, 
on  the  autopsy  must  rest  the  burden  of  proof  and  blame.  When  we 
realize  how  small  a  lesion  may  produce  hypersensitiveness,  it  will  readily 
be  understood  how  easily  the  clinician  and  pathologist  may  fail  to  detect 
the  lesion. 

A  large  part  of  our  knowledge  regarding  the  specificity  of  the  tu- 
berculin reaction  has  been  gained  from  veterinary  practice,  as  the  results 
of  a  test  in  an  animal  could  immediately  be  controlled  by  the  autopsy 
findings.  Thus  Fraenkel1  collected  from  the  literature  8000  carefully 
observed  instances,  and  found  only  from  2  to  3  per  cent,  of  differences 
between  the  result  of  the  tuberculin  test  and  of  the  autopsy.  Voges,2 
in  7327  instances,  noted  2.7  per  cent,  of  contradictions.  Kuhnau,3 
Bang,4  and  von  Behring 5  speak  of  similar  experiences. 

It  has  long  been  known  that  the  prevalence  of  tuberculous  findings 
anatomically  far  exceeded  the  number  of  cases  recognized  clinically. 
Among  cattle,  anatomic  tuberculosis  is  found  in  from  12  to  25  per 
cent.,  and  about  80  per  cent,  or  more  react  to  tuberculin.  In  many  of 
the  latter,  however,  the  disease  does  not  progress,  but,  on  the  contrary, 
tends  to  recede. 

Similar  conditions  exist  in  human  pathology.  That  tuberculosis  is 
very  frequent  among  adults  is  now  well  known.  The  figures  of  Nageli 6 
and  Burkhardt7  showed  that  the  increasing  frequency  of  tuberculous 
infection  with  advancing  years  reached  over  90  per  cent,  among  those 
past  the  eighteenth  year;  these  figures  are  now  well  corroborated.  Ham- 
burger,8 in  the  published  results  of  an  analysis  of  848  autopsies  on  chil- 
dren, showed  that  tuberculosis  was  in  evidence  in  40  per  cent.,  increasing 

1  Zeitschr.  f.  Tuberk.,  1900,  i,  291. 

2  "Tuberculin  und  Organismus,"  Jena,  1905,  77. 

3  Berl.  tierarztl.  Wochenschr.,  1899,  78. 

4  Sixth  Intel-nation.  Congress  on  Tuberculosis,  1908,  211. 

5  Beit.  z.  exper.  Therap.,  1905,  x,  1. 

6  Virch.  Arch,  f .  path.  Anat.,  1900,  clx,  426. 

7  Zeitschr.  f .  Hyg.  u.  Infectionsk.,  1906,  liii,  139. 
s  Wien.  klin.  Wochenschr.,  1907,  xx,  1070. 

40 


626      ANAPHYLAXIS    IN    RELATION   TO    INFECTION    AND    IMMUNITY 

from  4  per  cent,  among  infants  under  three  months  of  age  to  70  per  cent, 
among  children  from  eleven  to  fourteen  years.  This  explains,  in  part 
at  least,  the  relatively  high  resistance  of  children  to  tuberculin,  the 
difficulty  there  is  said  to  be  in  eliciting  reactions,  and  the  necessity  that 
exists  for  using  large  doses.  Usually,  when  children  fail  to  react,  it  is 
because  they  are  not  tuberculous  or  because  the  lesion  is  too  small, 
whereas  in  later  years,  until  adult  life  is  reached,  the  reaction  is  observed 
with  increasing  frequency  and  with  smaller  doses,  because  the  incidence 
of  infection  increases  progressively  from  5  per  cent,  during  infancy  to 
90  per  cent,  and  over  in  adult  life. 

The  prevalence  of  tuberculosis,  however,  by  no  means  indicates  that 
the  infected  individual  suffers  ill  health  or  will  succumb  to  the  infection. 
An  individual  may  be  enjoying  excellent  health,  and  still  harbor  a  tu- 
berculous lesion,  and  display  a  marked  degree  of  hypersensitiveness  to 
tuberculin.  Such  a  person  is  not  usually  regarded  as  tuberculous  until 
there  are  tangible  symptoms  referable  to  its  existence.  It  is  important 
to  remember  that  tuberculin  is  an  index  of  tuberculous  infection,  and  not  of 
disease  in  a  clinical  sense.  Numbers  of  persons  and  cattle  reacting  to 
tuberculin  remain  healthy  and  do  not  develop  symptoms  of  disease,  the 
autopsy  disclosing  the  presence  of  inactive  or  regressing  lesions. 

In  former  years  it  was  considered  possible  to  obtain  false  positive 
reactions  in  convalescents  and  patients  in  an  enfeebled  condition  who 
were  non-tuberculous,  and  also  in  other  diseases,  such  as  syphilis, 
leprosy,  and  actinomycosis.  More  accurate  anatomic  statistics  and 
careful  studies  of  the  tuberculin  test  administered  to  a  large  number  of 
individuals,  healthy,  tuberculous,  and  sufferers  from  other  diseases, 
have  gradually  changed  the  attitude  of  the  profession  and  served  to 
establish  the  high  specificity  of  the  tuberculin  reaction. 

Sources  of  Error  in  the  Tuberculin  Reaction. — From  the  foregoing 
it  will  readily  be  understood  that  most  errors  in  the  tuberculin  reaction 
refer  to  false  negative  rather  than  to  false  positive  reactions. 

False  positive  reactions  may  be  observed  in  leprosy,  where  the  bacillus 
bears  such  close  morphologic  and  biologic  resemblance  to  the  tubercle 
bacillus,  and  it  is  likewise  true  that  massive  doses  of  tuberculin  injected 
subcutaneously  may  produce  a  toxic  fever  in  debilitated  individuals, 
but  positive  reactions  in  healthy  individuals  can  usually  be  ascribed — 
(a)  to  a  small  hidden  tuberculous  lesion  or  (b)  to  a  healed  tuberculous 
lesion.  As  just  stated,  tuberculin  simply  indicates  hypersensitiveness  to 
the  tubercle  protein,  and  this  may  exist  with  a  very  small  unimportant 
lesion,  or  persist  after  a  lesion  has  been  " healed"  to  the  extent  of  en- 
capsulation. 


TUBERCULIN    REACTION  627 

False  negative  reactions  are  much  more  likely  to  occur,  and  the  various 
conditions  that  may  be  responsible  for  these  should  be  well  understood 
and  remembered. 

1.  In  the  final  stage  of  tuberculosis,  especially  in  miliary  tuberculosis 
and  in  tuberculous  cachexia,  as  in  the  third  stage  of  pulmonary  tubercu- 
losis, the  tuberculin  reaction  may  be  negative,  or  be  attained  only  after 
the  injection  of  very  large  doses.     There  is  a  lessened  cutaneous  re- 
activity (cachectic  reaction),  marked  by  the  appearance  of  colorless  or 
pinkish  spots,  instead  of  an  intense  papillary  eruption.     Koch  and  Ehr- 
lich  have  explained  this  by  assuming  that  the  tissues  had  become  too 
thoroughly  saturated  with  tuberculin  produced  at  the  infected  area  to 
respond  to  further  artificial  additions.     This  condition  may  be  regarded 
as  analogous  to  a  state  of  anti-anaphylaxis  in  which  we  may  consider 
the  free  and  sessile  receptors  united  with  the  tubercle  protein  or  the 
cells  loaded  with  the  antigen,  with  depression  of  cellular  activity. 

2.  In  the  first  stage  of  infection.     At  this  period  the  antibody  has  not 
been  formed  in  sufficient  amounts,  different  authors  obtaining  such 
findings  in  nurslings. 

3.  In  small,  completely  healed  lesions,  especially  in  those  showing 
nothing  but  scar  tissue,  as  in  the  apex  of  a  lung.     In  these  the  antibody 
and  cellular  hypersensitiveness  have  disappeared,  and  while  the  lesion 
may  have  been  tuberculous,  one  cannot  tell  anatomically,  in  a  given 
instance,  whether  or  not  hypersensitiveness  should  have  been  present. 

4.  During  continued  treatment  with  tuberculin,  when  the  reaction 
may  be  negative  owing  to  a  condition  of  anti-anaphylaxis. 

5.  During  measles.     As  von  Pirquet  and  Preisich  have  demonstrated, 
the  cutaneous  reactivity  disappears  during  the  first  days  after  the  erup- 
tion, reappearing  after  about  a  week.     Greuner  showed  that  the  lt  Stick- 
reaktion"  which  occurs  after  large  doses  of  tuberculin  did  not  disappear 
entirely,  indicating  that  in  measles  there  is  a  lessened  activity,  rather 
than  a  total  disappearance. 

6.  Finally,  according  to  von  Pirquet,  there  are  a  few  cases  in  which 
we  have  a  minimal  reactivity  and  to  which  none  of  the  former  explana- 
tions can  be  applied.     Some  cases  of  active  tuberculosis  may  show  only 
a  slight  hypersensitiveness,  although  they  are  not  cachectic. 

Of  course,  it  can  readily  be  understood  that  the  use  of  weak  or  an 
otherwise  unsatisfactory  solution  of  tuberculin  and  an  improper  dosage 
and  technic  will  greatly  influence  the  results.  Likewise  errors  in  inter- 
preting what  constitutes  a  positive  tuberculin  reaction  are  to  be  guarded 
against.  This  applies  especially  to  veterinary  practice.  For  example, 
cattle  brought  from  the  fields  and  confined  in  a  stall  for  the  purpose  of 


628      ANAPHYLAXIS   IN   RELATION   TO    INFECTION   AND   IMMUNITY 

making  a  tuberculin  test  may  exhibit  a  fever  for  several  days  without 
apparent  cause.  On  the  other  hand,  dishonest  dealers  may  force  cattle 
to  drink  cold  water  or  have  given  cold  water  irrigations  just  before  the 
temperature  is  taken,  preventing  the  registration  of  a  febrile  reaction. 

Methods  of  Conducting  the  Tuberculin  Test. — The  object  of  the 
tuberculin  test  is  to  introduce  sufficient  tubercle  protein  to  react  with 
the  tubercle  antibody,  with  the  formation  of  the  protein  poison,  which 
shows  its  presence  and  effects  by  a  general,  a  local,  or  a  focal  reaction  or 
by  a  combination  of  these.  Various  methods  have  been  proposed,  of 
which  the  following  are  best  known: 

1.  The  subcutaneous  tuberculin  test  is  the  oldest  test  of  its  kind,  having 
been  discovered  by  Koch1  in  1891.     It  consists  in  the  subcutaneous 
injection  of  old  tuberculin.     A  positive  reaction  manifests  itself  in  a 
constitutional  disturbance,  accompanied  by  fever,  a  local  reaction  at  the 
site  of  injection  ("Stichreaction")?  and  frequently  a  focal  reaction  at  the 
site  of  tuberculous  disease. 

2.  The  cutaneous  tuberculin  test  of  von  Pirquet,2  consisting  in  the 
local  application  of  old  tuberculin  to  a  superficial  abrasion  of  the  skin. 
A  positive  reaction  is  indicated  by  redness,  edema,  and  other  inflamma- 
tory phenomena. 

3.  The  conjunctival  tuberculin  test  of  Wolff-Eisner3  and  Calmette,4 
consisting  in  the  local  application  to  the  conjunctiva  of  one  eye  of  a  drop 
of  1  per  cent,  solution  of  old  tuberculin  or  purified  tuberculin.     A  positive 
reaction  is  indicated  by  congestion  and  lacrimation. 

4.  The  percutaneous  tuberculin  test  of  Moro  and  Doganoff,5  consisting 
in  the  application  of  tuberculin  ointment  prepared  by  mixing  equal  parts 
of  old  tuberculin  and  anhydrous  lanolin  and  applying  it  to  the  skin  over 
the  upper  portion  of  the  abdomen  or  about  the  nipple.     A  positive  reac- 
tion is  indicated  by  an  efflorescence  of  papules  upon  the  anointed  skin. 

5.  The  intracutaneous  tuberculin    test  of   Mendel6  and   Mantoux,7 
consisting  in  injecting  into  the  superficial  layers  of  the  skin  0.05  c.c.  of 
diluted  old  tuberculin.     A  positive  reaction  is  denoted  by  infiltration 
and  hyperemia  about  the  site  of  injection,  similar  to  the  reaction  to  the 
cutaneous  test. 

Comparative  Delicacy  and  Relation  of  the  Various  Tuberculin  Tests. 
— In  judging  of  the  comparative  delicacy  of  the  various  tuberculin 

iDeut.  med.  Wochenschr.,  1891,  xvii,  101. 

2  Berl.  klin.  Wochenschr.,  1907.  xliv,  699. 

3  Discussion,  Berl.  klin.  Wochenschr.,  1907,  xliv,  70. 

4Presse  me'dicale,  1907,  xv,  388.          5  Wien.  klin.  Wochenschr..  xx,  933. 

• Med.  Klin.,  1908,  iv,  402.  7  Munch,  med.  Wochenschr.,  1908,  No.  40. 


TUBERCULIN    REACTION  629 

tests  by  a  review  of  the  literature,  it  must  be  remembered  that  results 
will  vary  according  to  the  portion  of  the  body  inoculated,  as  sensitive- 
ness of  the  cells  varies  in  different  parts  of  the  body,  and  there  are  indi- 
vidual differences  in  various  persons  that  are  difficult  or  impossible  to 
explain. 

By  comparing  the  figures  obtained  as  the  result  of  different  tests 
upon  the  same  person  with  the  relative  frequency  of  individual  tests, 
Harnmah  and  Wolman1  have  drawn  the  following  conclusions: 

"1.  The  intracutaneous  and  subcutaneous  local  tests  are  the  most 
delicate  we  possess.  They  reveal  practically  the  full  percentage  of 
tuberculosis-infected  individuals. 

"2.  In  the  order  of  their  sensitiveness,  the  tests  arrange  themselves 
as  follows: 

Intracutaneous  Test. 
Subcutaneous  Local  Test. 
Cutaneous  Test. 
Subcutaneous  Test. 
Percutaneous  Test. 
Conjunctival  Test. 

"3.  There  is  a  definite  but  not  a  constant  relation  between  the  vari- 
ous tests.  An  individual  reacting  to  the  conjunctival  test  will,  as  a  rule, 
give  all  the  others,  but  not  always.  The  cutaneous  or  the  subcutaneous 
tests  may  be  negative  when  the  conjunctival  is  positive.  The  sub- 
cutaneous positive  when  the  cutaneous  is  negative,  etc.  Some  of  the 
unusual  variations  may,  no  doubt,  depend  upon  faulty  technic  in  per- 
forming the  tests,  but  all  can  certainly  not  be  thus  explained.  Local 
changes  in  sensitiveness  and  variation  in  the  facility  of  absorption  are 
probably  factors,  but  the  exact  conditions  are  not  understood. 

"4.  We  have  been  unsuccessful  in  an  attempt  to  make  the  cutaneous 
test  with  different  strengths  of  tuberculin  equivalent  to  the  conjunctival 
test." 

Although  the  subcutaneous  test  may  be  dangerous  on  account  of  the 
harm  that  may  result  from  too  severe  focal  reactions,  yet,  when  carefully 
conducted,  it  is  frequently  the  method  of  choice,  especially  in  the  diag- 
nosis of  an  obscure  pulmonary  lesion,  and  in  bone,  joint,  skin,  and  other 
local  infections,  where  the  focal  reaction  may  be  detected  by  direct 
examination.  It  is  to  be  emphasized,  however,  that  the  absence  of 
focal  changes  during  a  constitutional  reaction  does  not  exclude  the  tu- 
berculous nature  of  a  suspected  lesion.  In  children,  as  shown  by  Hamill, 
1  Tuberculin  in  Diagnosis  and  Treatment,  1912,  Appleton,  167. 


630      ANAPHYLAXIS    IN   RELATION   TO    INFECTION   AND    IMMUNITY 

Carpenter,  and  Cope,1  the  various  tuberculin  reactions  are  likely  to 
yield  results  that  are  quite  similar. 

The  Value  of  Tuberculin  in  Diagnosis. — As  previously  stated,  a  reac- 
tion to  tuberculin  means  essentially  that  the  individual  reacting  has  a  tu- 
berculous infection,  and  in  itself  means  nothing  more.  Since  tuberculin 
tests  disclose  inactive  and  relatively  benign  tuberculous  infections,  it 
has  little  value,  in  doubtful  cases,  in  aiding  us  to  decide  whether  or  not 
the  individual  has  active  disease,  which  clinically  is  the  type  of  infection 
about  which  we  are  most  concerned.  Lack  of  critical  discernment  in 
the  interpretation  of  the  reaction  and  its  apparent  indefiniteness  have 
contributed  toward  diminishing  its  diagnostic  value. 

A  positive  tuberculin  reaction  is  to  be  regarded  as  a  symptom,  or  as 
another  link  in  the  chain  of  clinical  evidence,  but  is  not  in  itself  indisputable 
evidence  that  a  certain  lesion  is  tuberculous,  for  it  can  never  decide  with 
certainty  an  otherwise  doubtful  diagnosis.  A  similar  example  is  that  of 
a  positive  Wassermann  reaction  in  a  patient  with  a  lesion  in  the  throat; 
such  a  reaction  does  not  necessarily  mean  that  the  lesion  is  syphilitic, 
for  the  lesion  itself  may  be  cancerous,  although,  coincidentally,  a  latent 
syphilitic  infection  may  be  present. 

If  tuberculin  could  differentiate  between  active  and  inactive  lesions 
according  to  the  degree  of  reaction,  its  value  would  be  greatly  increased. 
While  the  studies  of  Krompecker  and  Romer  upon  animals  indicate  that 
the  more  virulent  the  infection  the  greater  the  degree  of  hypersensitive- 
ness,  no  such  fixed  relation  exists  in  man.  All  that  may  be  said  is  that, 
in  general,  the  severer  the  reaction,  the  more  acute  the  infection.  On 
the  other  hand,  acute  miliary  tuberculosis  or  chronic  cachectic  cases 
may  react  negatively. 

The  conditions  under  which  a  negative  reaction  may  be  obtained  in  a 
tuberculous  person  are  to  be  carefully  borne  in  mind,  for  if  these  can  be 
excluded,  a  negative  tuberculin  reaction  precludes,  in  all  probability,  an 
active  or  clinically  important  tuberculous  lesion.  Tuberculin  has,  there- 
fore, a  higher  negative  than  a  positive  value  in  diagnosis. 

While  it  is  obviously  beyond  the  scope  of  this  volume  to  discuss  the 
diagnostic  value  of  tuberculin  in  tuberculous  infection  of  the  different 
organs,  I  may  briefly  refer  to  a  few  of  the  more  important  conclusions 
reached  by  individual  investigators  of  large  experience  in  this  particular 
field: 

1.  In  the  diagnosis  of  pulmonary  tuberculosis,  while  a  positive  consti- 
tutional or  local  tuberculin  reaction  is  never  conclusive  evidence  that  a 
iArchiv.  Int.  Medicine,  1908,  ii,  405. 


TUBERCULIN    REACTION  631 

definite  pulmonary  lesion  is  tuberculous,  a  focal  reaction,  on  the  other 
hand,  tells  definitely  of  the  presence  of  the  disease,  and  shows,  in  some 
measure  at  least,  its  extent.  In  these  questionable  cases,  therefore,  the 
subcutaneous  injection  of  tuberculin  finds  its  most  important  applica- 
tion, since  a  definite  focal  is  of  more  value  than  a  local  reaction. 

Tuberculin  may  also  be  of  service  when  the  symptoms  suggest  the 
presence  of  a  pulmonary  tuberculous  lesion,  but  the  physical  signs  are 
indefinite.  Here  the  focal  reaction  is  likely  to  be  slight  and  to  escape 
detection,  so  that  one  of  the  cutaneous  tests  are  usually  employed. 

2.  In  the  diagnosis  of  bone,  joint,  and  glandular  tuberculosis  the  sub- 
cutaneous test  is  likely  to  be  most  valuable,  on  account  of  the  focal  reac- 
tion of  hyperemia,  swelling,  heat,  and  pain  about  the  lesions.     The 
cutaneous  test  is  also  valuable,  but  since  this  may  react  on  account  of 
the  presence  of  a  lesion  situated  elsewhere,  the  focal  reaction  is  more 
conclusive.     According  to  Hamman  and  Wolman,  (a)  a  focal  reaction 
confirms  the  diagnosis  of  tuberculous  bone  or  joint  disease;    (b)  an  ab- 
sence of  reaction  to  the  subcutaneous  test  excludes,  with  the  highest 
probability,  the  presence  of  tuberculosis. 

3.  In  the  diagnosis  of  genito-urinary  and  pelvic  tuberculosis  a  positive 
tuberculin  reaction  is  of  little  value  unless  the  subcutaneous  method  is 
employed  and  the  physician  is  sure  of  his  ability  to  detect  a  focal  reac- 
tion, a  proceeding  that  may  be  very  difficult  or  indeed  impossible.     A 
positive  cutaneous  test  indicates  the  presence  of  a  lesion  somewhere 
in  the  body,  without  disclosing  the  nature  of  the  renal  or  pelvic  lesion. 
A  negative  reaction,  however,  is  of  more  value,  especially  when  the 
physician  bears  in  mind  the  conditions  under  which  a  falsely  negative 
result  may  occur. 

4.  In  the  diagnosis  of  tuberculosis  of  the  eye,  ear,  and  larynx,  the  tu- 
berculin reaction  usually  has  a  limited  value,  because  the  nature  of  the 
disease  can  be  so  readily  detected  by  direct  inspection.     In  tuberculosis 
of  the  larynx  a  focal  reaction  may  be  dangerous  on  account  of  edema. 
Similarly  in  advanced  tuberculosis  of  the  ear  a  focal  reaction  may  lead 
to  extension  of  the  process  to  the  meninges.     In  these  instances,  there- 
fore, a  cutaneous  test  should  first  be  made,  and  if  it  is  found  to  be  nega- 
tive or  inconclusive,  the  subcutaneous  test  should  be  applied  with  extra 
caution. 

5.  In  the  diagnosis  of  tuberculosis  of  the  skin  tuberculin  may  occasion- 
ally prove  of  value — especially  the  focal  reaction  following  subcutaneous 
injection  or  a  direct  local  application  upon  the  lesion  with  a  weak  dilu- 
tion, such  as  a  1  per  cent,  solution  of  old  tuberculin. 


632      ANAPHYLAXIS    IN    RELATION   TO    INFECTION   AND    IMMUNITY 

6.  In  the  diagnosis  of  tuberculosis  of  a  serous  membrane  tuberculin 
usually  possesses  a  limited  value.  In  tuberculous  meningitis  the  sub- 
cutaneous test  is  contraindicated,  as  a  focal  reaction  may  do  harm. 
Owing  to  the  acute  infection  the  cutaneous  test  may  be  negative,  and 
even  if  positive,  would  not  aid  greatly  in  the  diagnosis  because  the 
meningeal  condition  is  always  secondary  to  a  primary  focus.  Tubercu- 
lous pleurises,  dry  or  with  effusion,  and  unaccompanied  by  evident  pul- 
monary disease,  are  frequently  associated  with  a  low-grade  tuberculin 
hypersensitiveness.  According  to  Hamman  and  Wolman,  in  a  large 
proportion  of  cases  of  pleurisy  with  effusion  the  conjunctival  test  is 
negative  and  the  cutaneous  test  but  mildly  positive.  Bandelier  and 
Roepke  assert  that  in  a  dry  pleurisy  increased  pain  and  more  pro- 
nounced and  extensive  friction  may  occur  during  a  constitutional 
reaction  to  the  subcutaneous  test  and  indicate  a  focal  reaction.  In 
tuberculous  peritonitis  the  tuberculin  test  is  frequently  negative.  A 
positive  reaction  has  far  more  value,  especially  in  virulent  types  of  the 
disease,  which  come  on  insidiously  with  little  or  no  constitutional  dis- 
turbance. 

The  Value  of  Tuberculin  in  Prognosis. — As  previously  stated,  tu- 
berculin may  not  react  in  the  very  early  and  very  late  cases  of  tubercu- 
losis. In  patients  with  rapidly  advancing  lesions  the  power  to  react 
tends  to  decrease  and  frequently  is  absent.  But  this  condition  is 
apparent  without  the  aid  of  tuberculin.  While  Wolff-Eisner  and  Stadel- 
mann1  laid  some  stress  upon  the  conjunctival  reaction  in  prognosis, 
others  have  been  unable  to  confirm  the  results,  and,  as  stated  by  Ham- 
man and  Wolman,2  tuberculin  fails  to  yield  us  information  of  prog- 
nostic value  that  other  methods  of  clinical  observation  do  not  bestow. 

The  Dangers  of  Tuberculin. — Practically,  the  only  danger  lies  in 
the  subcutaneous  and  conjunctival  tests.  With  the  subcutaneous 
test,  the  greatest  danger  in  pulmonary  tuberculosis  is  the  possibility 
of  overdosage,  with  the  production  of  an  extensive  focal  reaction  which 
may  bring  on  hemorrhage  or  lead  to  local  extension  of  the  lesion.  Be- 
cause of  its  very  important  bearing  on  tuberculin  treatment,  the  sub- 
ject will  be  discussed  in  the  next  chapter.  The  same  danger  of  excessive 
focal  reaction  holds  for  tuberculous  meningitis  (increased  intracranial 
pressure),  in  tuberculous  laryngitis  (edema),  and  in  tuberculosis  of  the 
ear  and  nasal  accessory  sinuses  (extension  to  meninges) . 

The   cutaneous,    intracutaneous,    and   percutaneous   reactions   are 

1  Deutsch.  med.  Wochenschr.,  1908,  xxxiv,  180. 

2  "Tuberculin  in  Diagnosis  and  Treatment,"  1912,  179. 


TUBERCULIN    REACTION  633 

practically  devoid  of  danger,  providing  that  a  careful  technic  is  ob- 
served. 

Regarding  the  dangers  of  the  conjunctival  test,  opinions  differ. 
There  can  be  no  doubt,  however,  but  that  distressing  sequelae,  such  as 
severe  recurring  conjunctivitis,  phlyctenular  conjunctivitis,  and  corneal 
ulcers  with  permanent  opacities  have  resulted  from  the  use  of  this  test. 
Most  of  the  unfavorable  results  have  followed  instillation  in  already 
diseased  eyes,  or  of  too  strong  solutions,  but  this  is  not  true  of  all  cases. 
As  will  be  pointed  out  further  on,  in  the  first  instillation  not  over 
1  per  cent,  of  old  tuberculin  should  be  used,  and  a  second  instillation 
should  never  be  made  in  the  same  eye  for  at  least  several  years. 

Since  old  people  are  especially  prone  to  conjunctival  inflammation 
and  corneal  ulceration,  it  is  probably  better  to  exclude  them  from  the 
test.  Another  drawback  to  this  test  is  the  possibility  of  a  "flare  up" 
in  the  eye  following  subsequent  subcutaneous  administration  of  tuber- 
culin, either  for  diagnostic  or  therapeutic  purposes.  Hamman  and 
Wolman,  however,  do  not  consider  this  dangerous,  and  have  observed 
but  two  cases  in  an  extensive  experience. 

THE  SUBCUTANEOUS  TUBERCULIN  REACTION 

As  has  been  stated  repeatedly,  the  subcutaneous  injection  of  tu- 
berculin is  resorted  to  at  the  present  time  mainly  for  the  purpose  of 
eliciting  a  focal  reaction,  and,  as  a  rule,  this  is  more  easily  appreciable 
when  the  general  reaction  is  well  marked.  //  tuberculin  is  used  simply 
to  establish  whether  or  not  a  person  is  hypersensitive,  this  fact  may  be 
demonstrated  by  employing  much  smaller  doses,  as  by  the  cutaneous,  intra- 
cutaneous,  or  conjunctival  tests,  the  patient  being  spared  the  discomfort 
of  the  constitutional  symptoms. 

Variety  of  Tuberculin. — Koch's  old  tuberculin  is  now  used  almost 
exclusively.  The  technic  of  its  preparation  is  given  hi  the  chapter  on 
Tuberculin  Therapy. 

Manufacturing  chemists  usually  market  this  tuberculin  in  a  series 
of  dilutions,  labeled  and  accompanied  by  explicit  directions,  so  that  the 
physician  may  administer  practically  any  dose  desired  by  injecting 
so  many  minims  of  such  or  such  dilution.  Otherwise  a  series  of 
dilutions  are  readily  prepared  in  the  physician's  office  or  dispensary, 
according  to  the  method  followed  by  Hamman  and  Wolman: 

(a)  Seven  wide-mouthed  bottles  of  about  from  12  to  15  c.c.  ca- 
pacity, and  fitted  with  ground-glass  or  rubber  stoppers,  are  sterilized, 
labeled,  and  numbered  from  2  to  8. 


634      ANAPHYLAXIS    IN    RELATION   TO    INFECTION   AND    IMMUNITY 

(6)  The  diluent  is  sterile  0.8  per  cent,  salt  solution  with  0.25  per 
cent,  pure  phenol.  This  is  readily  prepared  by  adding  8  grams  of 
pure  sodium  chlorid  and  2.5  c.c.  of  pure  phenol  to  1000  c.c.  of  distilled 
water.  Dissolve,  and  filter  into  one  large  Erlenmeyer  flask  or,  prefer- 
ably, into  ten  smaller  flasks.  Sterilize  in  the  Arnold  sterilizer  for  one 
hour,  or  in  the  autoclave  for  twenty  minutes,  or  by  gently  boiling  for 
fifteen  minutes  on  each  of  two  consecutive  days. 

(c)  Into  each  bottle  place  9  c.c.  of  the  diluent  with  a  graduated  and 
sterile  pipet.     Bottle  1  contains  pure  tuberculin.     To  bottle  2  add  1 
c.c.  of  tuberculin  and  shake  carefully;   to  bottle  3,  1  c.c.  from  bottle  2 
and  shake;    to  bottle  4,  1  c.c.  from  bottle  3  and  shake;    continue  in 
this  manner  to  bottle  8,  from  which  1  c.c.  is  discarded. 

(d)  We  now  have  the  following  dilutions : 

No.  1 — pure  tuberculin. 

No.  2 — 0.1  c.c.  tuberculin  in  each  cubic  centimeter. 

No.  3—0.01  c.c.         " 

No.  4—0.001          c.c. 

No.  5—0.0001        c.c. 

No.  6—0.00001      c.c. 

No.  7— O.OQ0001    c.c. 

No.  8— O.OOQOOO,!  c.c. 

(e)  These  dilutions  are  usually  prepared  every  two  weeks.     When 
not  in  use,  the  bottles  are  kept  in  a  cool,  dark  place.     It  may  not  be 
necessary  to  prepare  all  dilutions.     For  example,  dilutions  No.  3  and 
No.  4  are  sufficient  for  diagnostic  purposes,  as  0.1  c.c.  of  No.  4  equals 
0.1  mg.  of  tuberculin  and  1  c.c.  of  No.  3  equals  10  mg.,  thus  affording 
an  ample  range  of  dosage. 

Method  of  Conducting  the  Test. — 1.  The  patient's  temperature  and 
pulse-rate  should  be  taken  every  two  hours  for  from  four  to  seven  days. 
This  is  easily  accomplished  in  a  hospital;  ambulatory  patients  can 
usually  be  readily  trained  to  take  their  own  temperature.  All  observa- 
tions should  be  recorded  in  writing,  and  preferably  on  a  temperature 
chart.  The  patient's  temperature  must  be  constantly  below  99°  F. 
before  beginning  the  test,  and,  if  necessary,  prolonged  rest  in  bed  should 
be  enforced  to  overcome  any  existing  fever.  The  test  may  be  given 
in  spite  of  a  daily  rise  of  not  over  100°  F.,  but  tuberculin  by  subcutaneous 
injection  should  be  given  only  exceptionally  to  febrile  patients. 

2.  A  very  careful  physical  examination  should  be  made,  and  the 
results  recorded  just  before  and  just  after  the  test  in  order  to  detect 
a  focal  reaction.     This  is  extremely  important,  for  it  is  our  main  justifi- 
cation for  injecting  the  tuberculin. 

3.  Injections  are  made  subcutaneously  in  the  region  of  the  back, 


TUBERCULIN    REACTION  635 

below  the  angle  of  the  scapula,  or  in  the  arm.  The  skin  needs  no  prep- 
aration other  than  to  be  rubbed  with  alcohol.  The  injections  are 
best  given  during  the  late  evening  hours  or  early  in  the  morning,  in 
order  that  the  temperature  and  pulse  changes  may  be  observed,  espe- 
cially twelve  hours  after  the  injection.  Records  of  temperature  and 
pulse  should  be  made  every  two  hours  during  the  day  and  night  for 
forty-eight  hours  following  an  injection.  Hamman  and  Wolman 
recommend  the  "Tuberculin  Sub  2  Syringe,"  made  by  the  Randall, 
Faichney  Co.  Each  syringe  should  be  sterilized  by  boiling  prior  to 
use,  and  it  is  recommended  that  a  separate  syringe  be  provided  for  each 
dilution. 

4.  Considerable  controversy  has  arisen  over  the  size  of  the  doses 
to  be  employed.  Koch's  directions  called  for  one  milligram  at  first, 
then  for  five,  then  for  ten,  and  if  no  reaction  followed  this  dose,  it  was 
repeated.  There  is  a  decided  tendency,  however,  to  use  smaller  doses. 
If  the  object  is  to  establish  the  presence  or  absence  of  tuberculin  hyper- 
sensitiveness,  mild  reactions  will  suffice,  and  for  this  purpose  small 
doses  repeated  or  in  gradually  increasing  amounts  may  be  given.  If 
one  aims  to  produce  a  focal  reaction,  larger  doses  and  more  rapid  in- 
crease are  desirable.  Hamman  and  Wolman  advise  the  following  plan 
for  adults :  For  the  first  dose,  -J-  mg.  is  given.  If  there  is  a  slight  febrile 
reaction  of  about  one-half  a  degree,  and  especially  if  this  is  accompanied 
by  a  local  reaction  at  the  point  of  injection,  the  second  dose,  which 
is  the  same  as  the  first,  is  given  at  the  end  of  forty-eight  hours.  The 
reaction  will  now  most  likely  be  more  conclusive.  If  there  is  no  appre- 
ciable reaction  after  the  first  dose,  the  second,  consisting  of  one  milli- 
gram, is  given  at  the  end  of  forty-eight  hours,  and  the  third  dose,  if 
one  is  necessary,  consists  of  five  milligrams.  Occasionally  the  third 
dose  must  be  repeated,  or  even  ten  milligrams  given  if  the  negative  result 
is  at  variance  with  the  clinical  impressions. 

If  the  temperature  shows  any  irregularities,  the  intervals  between 
injections  should  be  prolonged  to  three  or  four  days  or  more. 

Roth-Schultz  advise  the  following  doses:  0.5  mg.;  1.25  mg.,  and 
2.5  mg.  as  the  terminal  dose. 

For  children  under  fifteen  years  of  age  smaller  doses  are  indicated — 
an  initial  dose  of  one-tenth  milligram  and  a  terminal  dose  of  one  milli- 
gram, with  one  or  two  intervening  doses.  Baldwin  has  advised  0.05, 
0.2,  0.5,  and  1  mg. 

The  Reaction. — The  constitutional  reaction  is  quite  variable.  Fever 
is  the  most  delicate  indicator.  A  rise  of  1°  F.  or  more  above  the  previ- 
ous maximum  is  considered  positive,  especially  if  it  is  accompanied  by  a 


636      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

local  and  a  focal  reaction.  A  definite  febrile  reaction  due  to  tuberculin 
is  rare  without  the  presence  of  a  local  reaction  to  the  same  or  the  pre- 
ceding doses.  General  symptoms  of  headache,  muscle  pains,  anorexia, 
nausea,  etc.,  may  accompany  the  reactions.  The  local  reaction  consists 
of  redness  and  pain  at  the  site  of  injection,  with  tenderness  of  the  neigh- 
boring lymph-glands,  and  is  absolutely  specific  of  tuberculin  hyper- 
sensitiveness.  The  focal  reaction  consists  of  an  inflammatory  reaction 
with  the  production  of  rales,  change  in  breath-sounds,  etc. 

THE  INTRACUTANEOUS  TUBERCULIN  REACTION 

Variety  of  Tuberculin. — Koch's  old  tuberculin  is  used  either  in  one 
dose  of  0.005  mg.,  or  preferably  in  three  different  doses  injected  simul- 
taneously; these  will  be  described  further  on. 

Method  of  Conducting  the  Test. — The  skin  of  the  forearm  is  cleansed 
with  alcohol  and  then  dried.  A  small  glass  syringe  fitted  with  a  fine 
needle  is  used.  A  separate  syringe  is  used  for  the  control  fluid,  con- 
sisting of  sterile  salt  solution,  and  three  others  for  each  of  the  different 
dilutions  used.  In  performing  this  test  Hamman  and  Wolman  make 
four  simultaneous  injections: 

First:  0.05  c.c.  of  sterile  normal  salt  solution  (control). 

Second:    0.05  c.c.  of  a   1  :  1,000,000  dilution  of  old  tuberculin, 

which  equals  0.00005  mg.     This  equals  a  dose  of  0.05  c.c.  of 

dilution  No.  4,  just  described  in  the  preceding  test. 
Third:  0.05  c.c.  of  a  1  : 100,000  dilution,  or  0.05  c.c.  of  dilution  No. 

3,  equivalent  to  0.0005  mg. 
Fourth:  0.05  c.c.  of  a  1:10,000  dilution,  or  0.05  c.c.  of  dilution  No. 

2,  equivalent  to  0.0005  mg. 

Mantoux  uses  the  last  or  fourth  dose  only.  The  injections  are  given 
with  the  skin  held  taut  or  pinched  up  in  a  fold  between  the  index-finger 
and  thumb.  The  needle  is  inserted  superficially,  with  the  aperture 
directed  toward  the  outer  surface  of  the  skin.  If  the  point  of  the  needle 
is  in  the  skin,  a  wiiite  elevation  occurs  immediately  upon  the  introduc- 
tion of  the  solution;  if  it  is  in  the  subcutaneous  tissue,  no  infiltration  is 
apparent.  Cohen1  injects  T.  R.,  the  first  doses  being  one  ten-millionth, 
one  millionth,  and  one  hundred  thousandth  of  a  milligram.  If  these 
injections  provoke  no  reactions,  he  repeats  the  tests  with  doses  ranging 
as  high  as  ten  milligrams. 

The  Reaction. — The  reaction  consists  of  infiltration  and  hyperemia 
about  the  site  of  injection  similar  to  the  reaction  in  the  cutaneous  test. 
It  appears  in  from  six  to  eight  hours,  reaches  its  maximum  intensity 
1  Jour.  Infect.  Dis.,  1917,  xx,  233. 


TUBERCULIN    REACTION  637 

in  from  twenty-four  to  forty-eight  hours,  and  usually  disappears  in 
from  six  to  ten  days.  The  salt  solution  generally  produces  a  trau- 
matic reaction,  similar  to  a  mild  tuberculin  reaction,  which  subsides 
in  forty-eight  hours. 

The  reactions  are  best  recorded  after  twenty-four  hours,  and  the 
simplest  method  of  recording  the  results  is  to  measure  the  width  of  the 
area  of  infiltration  of  each  reacting  point. 

THE  CUTANEOUS  TUBERCULIN  REACTION  (VON  PIRQUET) 
Variety  of  Tuberculin. — Undiluted  old  tuberculin  is  now  used  almost 
exclusively  in  conducting  the  test. 

Method  of  Conducting  the  Test. — 1.  The  flexor  surface  of  the  fore- 
arm is  chiefly  used  for  making  the  applications,  but  it  should  be  re- 
membered that  tests  performed  on  different  portions  of  the  body  are 
not  strictly  comparable. 

2.  The  skin  is  cleansed  lightly  with  alcohol  and  dried.     Three  abra- 
sions are  made,  about  lJ/£  or  2  inches  apart,  with  a  von  Pirquet  skin 
borer  (Fig.  126)  or  with  a  needle,  small  lancet,  or  blood  sticker.     The 
object  is  to  scarify  the  superficial  layers  of  the  skin,  avoiding  as  much  as 
possible  bleeding,  although  a  few  small  points  of  blood  should  appear. 
To  the  upper  and  lower  abrasions  add  a  drop  of  tuberculin;   after  ten 
minutes  wipe  away  the  excess  with  a  bit  of  cotton.     No  shield  or  pro- 
tective dressings  are  required.     The  middle  abrasion  is  the  control, 
and  shows  the  amount  of  traumatic  reaction  following  the  scarifying 
process.    Due  precautions  should,  of  course,  be  observed  that  none  of 
the  tuberculin  flows  down  the  arm  and  reaches  this  spot. 

3.  The  tests  are  inspected  at  the  end  of  twenty-four  hours. 

The  Reaction. — The  traumatic  reaction  as  shown  in  the  control 
area  may  present  an  inflammatory  areola  with,  at  times,  slight  infiltra- 
tion. Before  a  test  may  be  considered  positive  its  areola  should  be  at 
least  five  millimeters  wider  than  the  control  area.  The  reactions  are 
usually  designated  as  follows: 

1.  Negative  Reaction. — No  appreciable  difference  between  the  tu- 
berculin areas  and  the  control. 

2.  Slight  Reaction. — Definite  but  slight  redness  with  some  infiltration. 

3.  +  Reaction. — A  wider  area  of  redness,  with  definitely  raised 
centers. 

4.  ++  Reaction. — Wider  area  of  redness,  with  more  marked  in- 
filtration than  +. 

5.  +  +  +  Reaction. — Unusual  redness  and  a  wide  area  of  infiltra- 
tion, all  cases  which  go  on  to  vesiculation. 


638      ANAPHYLAXIS    IN    RELATION    TO    INFECTION    AND    IMMUNITY 

The  usual  or  normal  reaction  begins  to  appear  in  from  four  to  six 
hours,  reaches  its  maximum  intensity  in  from  twenty-four  to  forty- 
eight  hours,  and  then  fades  rapidly,  although  the  infiltration  may  persist 
for  some  days.  Special  types  of  the  reaction  have  been  described  as 
follows: 


FIG.  126. — METHOD  OF  PERFORMING  A  VON  PIRQUET  TUBERCULIN  TEST. 

The  abrasion  is  being  made  over  the  insertion  of  the  deltoid  muscle.  The 
borer  is  held  firmly  and  perpendicular  to  the  arm.  A  quick  rotatory  motion  serves 
to  remove  a  circular  area  of  epidermis. 

The  barer  is  shown  in  the  upper  right-hand  corner. 

1.  The  premature  reaction,  characterized  by  a  rapid  course  and  slight 
intensity.     This  type  is  supposed  to  occur  in  patients  with  manifest 
tuberculosis  who  are  not  doing  well. 

2.  The  persisting  reaction,  which  reaches  its  maximum  intensity 
about  the  second  day  and  persists  for  a  week  or  longer. 


FIG.  127. — A  POSITIVE  CUTANEOUS  TUBERCULIN  REACTION  (VON  PIRQUET). 

Child  with  incipient  pulmonary  tuberculosis;  a  +  reaction.     The  control  scarification 

is  barely  to  be  seen,  and  is  midway  between  the  tuberculin  reactions. 


FIG.   128. — A   POSITIVE  CONJUNCTIVAL  TUBEHCULIN    REACTION  (WOLFF-EISNER- 

CALMETTE). 

Severe  reaction  (H — | — h)  in  a  tuberculous   cow;    appearance  of  the  eye  about 
fourteen  hours  after  second  instillation  of  tuberculin. 


TUBERCULIN   REACTION  639 

3.  The  late  reaction,  which  appears  after  twenty-four  hours  and  de- 
velops and  recedes  slowly.    These  last  two  types  are  believed  to  occur 
in  patients  having  inactive  lesions. 

4.  The  cachectic  reaction,  which  is  characterized  by  infiltration  with 
little  or  no  redness.     This  type  is  common  in  the  late  stages  of  tubercu- 
losis. 

5.  The  scrofulous  reaction,  which  is  characterized  by  numerous  small 
elevated  nodules,  which  may  also  appear  on  the  extremities  and  trunk. 
This  reaction  is  peculiar  to  children  and  rare  in  adults. 

THE  CONJUNCTIVAL  TUBERCULIN  REACTION  (CALMETTE) 

Variety  of  Tuberculin  Used. — A  1  per  cent,  solution  of  Koch's  old 
tuberculin  is  now  generally  used,  as  it  is  quite  reliable,  least  expensive, 
and  the  results  that  follow  its  use  are  more  regular  than  those  ob- 
tained with  purified  tuberculin  (0.5  to  2  per  cent,  aqueous  solution). 

Method  of  Conducting  the  Test — The  conjunctive  of  both  eyes 
are  inspected  to  ascertain  if  there  is  any  evidence  of  disease  and  if  they 
are  strictly  comparable  in  color.  The  lower  lid  of  one  eye  is  drawn  for- 
ward to  form  a  little  pouch,  and  the  patient  is  directed  to  look  upward; 
one  drop  of  a  1  per  cent,  dilution  of  old  tuberculin  is  then  applied  from 
an  ordinary  eye-dropper  to  the  lid  at  the  inner  canthus.  Profuse 
lacrimation  impairs  the  test,  and  it  is  useless  to  attempt  it  with  weeping 
or  resisting  children.  If  no  reaction  is  apparent  and  it  is  still  desired 
to  further  the  test,  a  drop  of  a  5  per  cent,  dilution  may  be  placed  in  the 
opposite  eye. 

The  Reaction. — In  a  positive  reaction  the  conjunctiva  begins  to 
redden  in  from  six  to  eight  hours,  and  reaches  its  maximum  in  from 
twenty-four  to  forty-eight  hours,  and  then  rapidly  subsides  and  dis- 
appears in  from  four  to  six  days.  In  mild  reactions  the  inner  canthus 
is  the  seat  of  the  most  marked  changes.  Positive  reactions  have  been 
classified  as  follows: 

+  Reaction:   Definite  palpebral  redness. 

+  +  Reaction :  More  marked  palpebral  redness  with  secretion. 

-f-f+  Reaction:  Palpebral  and  bulbar  redness  with  subjective 
symptoms  and  well-marked  secretion.  (See  Fig.  128). 

Precautions. — On  account  of  severe  reactions  and  the  danger  of 
inflicting  permanent  injury  on  the  cornea  there  is  a  growing  tendency 
to  regard  this  reaction  with  disfavor.  Hamman  and  Wolman,  however, 
believe  that,  with  proper  precautions,  these  risks  may  be  minimized, 
if  not  completely  avoided;  they  believe,  moreover,  that  the  risk  in- 


640      ANAPHYLAXIS    IN    RELATION   TO    INFECTION    AND    IMMUNITY 

curred  is  not  great  enough  to  warrant  the  abandonment  of  a  procedure 
that  has  proved  itself  of  such  great  value  in  diagnosis.  Nevertheless, 
they  emphasize  the  necessity  for  observing  proper  precautions,  and 
give  the  following  rules  to  be  adopted: 

1.  The  conjunctival  test  should  never  be  repeated  in  the  same  eye. 

2.  A  solution  stronger  than  1  per  cent,  original  tuberculin  should 
never  be  used  for  making  the  first  instillation. 

3.  Any  existing  inflammatory  disease  of  the  eye  is  an  absolute  con- 
traindication to  the  test. 

4.  The  test  should  not  be  given  to  manifestly  scrofulous  children. 

5.  Skin  diseases  in  which  the  lesions  are  situated  upon  the  face,  near 
the  eye,  especially  when  these  are  suspected  of  being  tuberculous,  pre- 
clude the  application  of  the  test. 

6.  It  is  safest  not  to  give  the  test  to  elderly  persons,  and  particularly 
to  arteriosclerotics,  as  they  are  unduly  prone  to  develop  corneal  ul- 
ceration. 

THE  PERCUTANEOUS  TUBERCULIN  REACTION  (MoRO) 
Variety  of  Tuberculin  Used. — This  consists  of  5  c.c.  of  old  tuberculin 
and  5  grams  of  anhydrous  lanolin  thoroughly  mixed.  The  ointment 
retains  its  potency  for  many  months  when  preserved  in  a  cold  dark 
place.  Manufacturers  market  the  preparation  in  small  collapsible 
tubes  containing  sufficient  for  at  least  one  or  two  tests. 

Method  of  Conducting  the  Test. — A  piece  of  ointment  about  the 
size  of  a  pea  is  thoroughly  rubbed  for  at  least  a  minute  into  an  area  of 
skin  about  two  inches  in  diameter  over  the  upper  abdomen  or  near  a 
nipple.  The  patient  may  be  instructed  how  to  apply  this  ointment,  or 
the  physician  may  do  it  himself,  protecting  the  finger  with  a  rubber  cot. 
The  Reaction. — This  may  appear  within  twenty-four  hours,  or  be 
delayed  for  as  long  as  from  four  to  six  days.  It  consists  of  an  eruption 
of  slightly  elevated  papules  situated  upon  a  hyperemic  base,  which 
vary  in  size  from  a  pinhead  to  large  areas  of  infiltration  (Fig.  129).  It 
subsides  in  from  three  to  ten  days.  Moro  described  these  grades  of 
reaction  as  follows: 

1.  Mild  reactions:  From  1  to  10  scattered  papules. 

2.  Moderate  reactions:    About  50  or  more  papules,  partly  discrete, 
partly  confluent,  and  with  a  general  reddening  of  the  skin.     There  may 
be  well-marked  itching. 

3.  Severe   reactions:     Numerous   large,    extremely   red   papules   or 
vesicles  up  to  8  mm.  in  diameter,  having  an  intensely  reddened  base. 


FIG.  129. — A  POSITIVE  PERCUTANEOUS  TUBERCULIN  REACTION  (MORO). 

E.  McK.,  adult  male  with  arrested  early  pulmonary  tuberculosis.     Lesions  about 

sixty  hours  after  application  of  tuberculin  ointment. 


TUBERCULIN    REACTION  641 

TUBERCULIN  REACTIONS  AMONG  THE  LOWER  ANIMALS 

In  veterinary  practice  tuberculin  is  used  almost  solely  for  diagnostic 
and  only  occasionally  for  therapeutic  purposes. 

Four  reactions  are  in  common  use : 
The  Subcutaneous  Test. 
The  Conjunctival  Test. 
The  Cutaneous  Test. 
The  Intracutaneous  Test. 

The  technic  for  conducting  these  tests  and  the  reactions  secured  are 
quite  similar  to  those  just  described,  and  I  would  refer  the  veterinary 
surgeon  to  these  respective  descriptions.  Differences  in  technic  are 
confined  principally  to  dosage. 

The  Subcutaneous  Tuberculin  Test. — This  is  conducted  with  Koch's 
old  tuberculin.  The  method  of  preparation  is  given  in  the  succeeding 
chapter,  under  Tuberculin  Therapy.  Concentrated  tuberculin  is  pre- 
pared by  concentrating  the  bouillon  nitrate  to  one-tenth  its  original 
volume.  Diluted  tuberculin  is  the  concentrated  product  diluted  to  its 
original  volume  by  mixing  1  part  of  the  dilution  with  9  parts  of  0.5  to  1 
per  cent,  phenol  in  sterile  normal  salt  solution  or  distilled  water. 

The  injections  are  always  given  subcutaneously  hi  some  convenient 
area,  preferably  around  the  shoulder,  which  has  been  shaven  and  cleaned 
beforehand  with  a  solution  of  creolin. 

The  first  dose  for  horses  and  cattle  is  usually  0.4  c.c.  of  concentrated 
or  4  c.c.  of  diluted  tuberculin;  the  second  dose  is  usually  0.8  c.c.  of  con- 
centrated or  9  c.c.  of  the  diluted  tuberculin. 

The  general  local  and  focal  reactions  are  similar  to  those  previously 
described. 

The  Conjunctival  Tuberculin  Test. — For  this  test  Koch's  concen- 
trated tuberculin  may  be  used.  Preference  is  usually  given  to  the  puri- 
fied product,  prepared  as  follows:  Mix  one  part  of  Koch's  concentrated 
tuberculin  with  20  parts  of  absolute  alcohol.  The  precipitate  that  forms 
is  filtered  off  and  dried  over  sulphuric  acid.  This  powder  is  then  made 
up  into  a  4  per  cent,  and  8  per  cent,  solution  in  sterile  distilled  water. 

Two  or  three  drops  of  the  4  per  cent,  dilution  are  placed  in  the  inner 
canthus  of  one  eye,  to  sensitize  the  tissues.  After  twenty-four  hours, 
unless  a  positive  reaction  is  present,  two  or  three  drops  of  the  8  per 
cent,  solution  are  instilled  in  the  same  eye  in  the  same  manner.  The 
reaction  is  usually  apparent  in  from  six  to  twelve  hours  (Fig.  128). 

For  the  types  of  reaction  and  precautions  to  be  observed  see  the 
previous  description. 

4i 


642      ANAPHYLAXIS    IN    RELATION    TO    INFECTION   AND    IMMUNITY 

One  advantage  of  this  test  is  that  the  animal  will  give  a  reaction  in 
cases  where,  prior  to  the  test,  dishonest  dealers  have  injected  tuberculin. 

The  Cutaneous  Tuberculin  Test. — In  making  this  test  some  con- 
venient area  is  shaved  and  scraped  slightly  until  serum  exudes.  A  small 
amount  of  Koch's  old  tuberculin  is  applied  to  the  prepared  area.  In  a 
positive  case  a  well-marked  area  of  congestion  and  hyperemia  appear 
at  the  end  of  twenty-four  hours.  This  may  also  be  accompanied  by  a 
rise  in  temperature. 

The  Intracutaneous  Tuberculin  Test. — In  performing  this  test 
from  0.2  to  0.4  c.c.  of  Koch's  concentrated  tuberculin  are  injected  into 
the  skin  through  a  fine  needle.  A  white  swelling  should  appear  while 
the  injection  is  being  given;  if  it  does  not  appear,  the  injection  is  sub- 
cutaneous and  unsatisfactory  for  this  test.  The  appearance  of  hyper- 
emia and  redness  with  a  rise  in  temperature  indicates  a  positive  reaction. 


LUETIN  REACTION 

The  clinical  course  of  syphilis  indicates  that  the  infecting  micro- 
parasite,  Treponema  pallida,  possesses  all  the  qualities  essential  to  the 
development  of  an  anaphylactic  condition  in  syphilitic  patients.  Thus 
the  primary  lesion  appears  after  an  incubation  period  of  two  or  three 
weeks.  The  secondary  stage  is  manifested  by  periodic  waves  of  various 
general  symptoms;  the  primary,  secondary,  and  tertiary  lesions  show 
a  qualitative  difference.  Stimulated  by  von  Pirquet's  discovery  of  a 
specific  cutaneous  reaction  for  tuberculosis,  a  number  of  investigators 
(Finger  and  Landsteiner,  Wolff-Eisner,  Nobe,  Ciuffo,  Nicolas,  Favre, 
and  Gauthier)  attempted  to  obtain  a  specific  reaction  for  syphilis  by 
applying  extracts  of  syphilitic  tissues — prepared  from  syphilitic  fetal 
liver  or  chancre — to  the  skin  of  syphilitic  patients.  In  spite  of  some 
encouraging  effects  their  results  were,  on  the  whole,  contradictory. 
Further,  Neisser  and  Bruck  found  that  a  reaction  similar  to  that  pro- 
duced with  syphilitic  extract  can  be  obtained  also  with  a  concentrated 
extract  of  normal  liver.  This  peculiarity  of  the  skin  of  syphilitics  is 
ascribed  by  Neisser  to  what  he  calls  the  state  of  "  Umstimmung"  in  the 
later  stages  of  syphilis.  Both  Neisser  and  von  Pirquet  expressed  the 
hope  and  belief  that  a  reaction  may  be  secured  by  employing  an  extract 
of  pallida  free  from  tissue  constituents;  this  was  first  and  finally  ac- 
complished by  Noguchi,1  in  1911,  first  with  syphilitic  rabbits  and  then 
with  human  patients.  Noguchi  gave  the  appropriate  name  of  "luetin" 
1  Jour.  Exper.  Med.,  1911,  14,  557;  Jour.  Amer.  Med.  Assoc.,  Iviii,  1163. 


LUETIN   REACTION  643 

to  the  extract  of  pallida.  Theoretically,  one  should  not  expect  to 
obtain  an  allergic  reaction  in  syphilis  so  long  as  the  activity  of  pallida 
is  maintained  at  its  maximum,  or  in  the  very  early  stages,  before  there 
is  sufficient  time  for  antibody  formation.  One  can  reasonably  expect 
the  appearance  of  the  phenomenon  when  the  activity  of  the  micropara- 
site  begins  to  abate  through  a  gradually  acquired  defensive  power  of 
the  host,  or  under  an  effective  therapeusis,  as  in  the  later  stages  of  the 
disease  and  in  hereditary  syphilis.  Practical  results  have  borne  out 
these  theoretic  expectations. 

Preparation  of  Luetin. — At  least  six  different  strains  of  pallida  in  pure  cul- 
ture are  being  used  by  Noguchi  in  the  preparation  of  luetin.  These  are  cultivated 
in  ascites-kidney  agar  for  periods  of  six,  twelve,  twenty-four,  and  fifty  days,  at  37° 
C.,  under  anaerobic  conditions.  The  tubes  showing  large  numbers  of  spirochetes  are 
then  selected,  the  oil  is  poured  off,  the  tube  is  cut,  the  agar  column  is  removed,  and 
the  tissue  then  cut  off.  The  ascites-agar  cultures  are  then  carefully  ground  in  a 
sterile  mortar,  the  resulting  thick  paste  being  gradually  diluted  with  a  fluid  culture 
until  a  homogeneous  liquid  emulsion  is  secured.  The  preparation  is  next  heated 
for  an  hour  in  a  water-bath  at  60°  C.,  and  then  tricresol  or  phenol  added  to  make  0.5 
per  cent.  Cultures  are  made  from  this  suspension,  and  rabbits  inoculated  intra- 
testicularly ;  both,  after  suitable  intervals,  must  show  an  absolutely  sterile  preparation. 

The  luetin  should  be  kept  in  the  refrigerator  when  not  in  use.  The 
isolation  of  Treponema  pallidum  in  pure  culture  is  a  difficult  procedure, 
and,  obviously,  a  luetin  must  be  prepared  of  pure  cultures  and  from  as 
many  different  strains  as  possible.  While  pallida  quickly  loses  its  patho- 
genicity  in  artificial  culture-media  and  is  also  highly  susceptible  to  the 
influence  of  germicides,  its  preparation,  nevertheless,  is  an  important 
matter  requiring  skilful  supervision. 

A  control  fluid  prepared  of  sterile  agar  and  bouillon  in  exactly  the 
same  manner  as  luetin  was  originally  advised  by  Noguchi,  but  recently 
he  claims  that  its  use  is  not  necessary. 

Method  of  Application. — Luetin  is  not  applied  to  an  abrasion  of  the 
skin,  but  is  injected  intracutaneously  with  a  very  fine  needle  and  a  sterile 
syringe.  According  to  directions  from  the  Rockefeller  Institute,  the 
luetin  is  to  be  well  shaken,  diluted  with  an  equal  part  of  sterile  salt 
solution,  and  0.07  c.c.  injected  (0.035  c.c.  undiluted).  A  slightly 
smaller  dose,  as,  e.  </.,  0.05  c.c.,  may  be  used  for  children.  The  skin  of 
the  upper  arm  is  usually  selected  as  the  site  for  inoculation.  If  a 
control  fluid  is  used,  the  luetin  may  be  injected  into  the  skin  of  the  left 
and  the  control  fluid  into  the  skin  of  the  right  arm,  or  both  injections 
may  be  given  in  the  same  arm,  about  two  inches  apart,  the  control 
being  above  the  luetin.  As  shown  by  Sherrick,1  and  confirmed  by  my 
1  Jour.  Amer.  Med.  Assoc.,  1915,  July  31,  404. 


644      ANAPHYLAXIS    IN    RELATION   TO    INFECTION   AND    IMMUNITY 

associates  and  I,1  normal  and  non-syphilitic  persons  taking  iodids  or 
bromids  at  the  time  the  skin  test  is  made  or  while  these  drugs  are  still 
in  the  body  fluids  may  yield  well-marked  non-specific  reactions  which 
the  physician  may  readily  interpret  as  a  positive  luetin  reaction. 

After  cleansing  the  skin  with  alcohol  it  is  drawn  taut  or  pinched  up 
between  forefinger  and  thumb  and  the  needle  introduced,  with  the 
aperture  directed  toward  the  outer  surface  of  the  skin.  If  the  point 
of  the  needle  is  in  the  skin,  a  white  elevation  occurs  immediately  upon 
injecting  the  solution;  if  it  is  in  the  subcutaneous  tissue,  no  filtration 
is  apparent.  (See  Figs.  68  and  69.) 

A  special  tuberculin  syringe  may  be  used,  and  the  luetin  drawn 
directly  into  the  barrel  from  the  stock  bottle  and  diluted  with  sterile 
normal  salt  solution.  To  obviate  waste  and  the  likelihood  of  contamina- 
tion, I  dilute  a  portion  of  luetin  (1  c.c.)  with  an  equal  amount  of  sterile 
salt  solution  (1  c.c.)  in  a  sterile  vessel,  and  then  add  6  c.c.  more  of  sterile 
salt  solution,  shaking  thoroughly  and  placing  0.2  c.c.  in  each  of  30  small 
sterile  ampules,  which  are  then  sealed.  Before  using,  the  ampule  is 
shaken  thoroughly,  opened,  and  the  contents  aspirated  into  the  syringe; 
0.1  c.c.  is  allowed  for  waste  in  loading  the  syringe  and  displacing  air; 
0.1  c.c.  is  injected,  and  this  is  practically  equivalent  to  0.035  c.c.  of  the 
undiluted  luetin,  the  dose  recommended.  When  so  prepared,  the  dilu- 
tion keeps  well,  is  especially  adapted  for  dispensary  use,  and  the  phenol 
is  so  diluted  as  to  exclude  any  traumatic  reaction. 

Reactions. — Normal  or  Negative  Reactions. — In  the  majority  of 
normal  persons  the  injection  of  luetin  is  followed  by  a  very  slight  trau- 
matic reaction,  or  a  small  erythematous  area  appears,  after  twenty-four 
hours,  at  and  around  the  point  of  injection.  No  pain  or  itching  sensa- 
tion is  experienced;  the  reaction  recedes  in  forty-eight  hours  and  leaves 
no  induration.  In  certain  individuals  the  reaction  may  reach  a  stage 
of  small  papule  formation  after  from  twenty-four  to  forty-eight  hours, 
which  subsides  within  seventy-two  hours,  leaving  no  induration. 

Positive  reactions  have  been  classified  by  Noguchi  into  three  main 
varieties : 

(a)  Papular  Form. — "A  large,  raised,  reddish,  indurated  papule, 
usually  five  to  ten  millimeters  in  diameter,  makes  its  appearance  in 
twenty-four  to  forty-eight  hours.  The  papule  may  be  surrounded 
by  a  diffuse  zone  of  redness  and  show  marked  telangiectasis.  The 
dimensions  and  the  degree  of  induration  slowly  increase  during  the  fol- 
lowing three  or  four  days,  after  which  the  inflammatory  processes  begin 
to  recede.  The  color  of  the  papule  gradually  becomes  dark  bluish  red. 
1  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  718;  Jour.  Lab.  and  Clin.  Med.,  1917,  xi,  401. 


FIG.  130. — A  POSITIVE  LUETIN  REACTION. 

E.  C.,  adult  male;   tertiary  syphilis  with  detachment  of  the  retina;   papular  lesion 
of  moderate  severity;  about  forty-eight  hours  after  injection  of  0.07  c.c.  luetin. 


LUETIN   REACTION  645 

The  induration  disappears  within  one  week,  except  in  certain  instances 
in  which  a  trace  of  the  reaction  may  persist  for  a  longer  period.  The 
latter  effect  is  usually  met  with  among  cases  of  secondary  syphilis  under 
regular  mercurial  treatment  in  which  there  are  no  manifest  lesions  at 
the  time  of  making  the  skin  test.  Cases  of  congenital  syphilis  also  show 
this  reaction. 

"(6)  Pustular  Form. — The  beginning  and  course  of  this  reaction 
resemble  the  papular  form  until  about  the  fourth  or  fifth  day,  when  the 
inflammatory  processes  commence  to  progress.  The  surface  of  the 
indurated  round  papule  becomes  mildly  edematous,  and  multiple 
miliary  vesicles  occasionally  form.  At  the  same  time  a  beginning  cen- 
tral softening  of  the  papule  can  be  seen.  Within  the  next  twenty- 
four  hours  the  papule  changes  into  a  vesicle,  filled  at  first  with  a  semi- 
opaque  serum  that  later  becomes  definitely  purulent.  Soon  after  this 
the  pustule  ruptures  spontaneously  or  after  slight  friction  or  pressure. 
The  margin  of  the  broken  pustule  remains  indurated,  while  the  defect 
caused  by  the  escape  of  the  pustular  content  becomes  quickly  covered 
by  a  crust  that  falls  off  within  a  few  days.  About  this  time  the  indura- 
tion usually  disappears,  leaving  almost  no  scar  after  healing.  There 
is  a  wide  range  of  variation  in  the  degree  of  intensity  of  the  reaction 
described  in  different  cases,  as  some  show  rather  small  pustules,  while 
in  others  the  pustule  is  much  larger.  This  reaction  was  found  almost 
constantly  in  cases  of  tertiary  syphilis,  as  well  as  in  cases  of  secondary 
or  hereditary  syphilis  which  had  been  treated  with  salvarsan. 

"(c)  Torpid  Form. — In  rare  instances  the  injection  sites  fade  away 
almost  to  invisible  points  within  three  or  four  days,  so  that  they  may 
be  passed  over  as  negative  reactions.  But  sometimes  these  spots  sud- 
denly light  up  again  after  ten  days  or  even  longer  and  progress  to  small 
pustular  formation.  The  course  of  this  pustule  is  similar  to  that  de- 
scribed for  the  preceding  form. 

"This  form  of  reaction  has  been  observed  in  a  case  of  primary 
syphilis,  in  one  of  hereditary  syphilis,  and  in  two  cases  of  secondary 
syphilis,  all  being  under  mercurial  treatment." 

Aside  from  these  three  types  of  reactions,  there  have  since  been 
described  several  cases  of  the  formation  of  a  hemorrhagic  exudate,  the 
lesion  usually  rupturing  spontaneously  and  not  running  a  more  severe 
or  a  longer  course  than  the  pustular.  Two  such  reactions  have  been 
reported  by  Kilgore.1 

"  Neither  in  syphilitics  nor  in  parasyphilitics  did  a  marked  consti- 
tutional effect  follow  the  intradermic  inoculation  of  luetin.  In  most 
1  Jour.  Amer.  Med.  Assoc.,  Ixii,  1236. 


646      ANAPHYLAXIS   IN   RELATION   TO   INFECTION   AND   IMMUNITY 

positive  cases  a  slight  rise  in  temperature  took  place,  lasting  for  one  day. 
In  three  tertiary  cases  and  in  one  hereditary  case,  however,  general 
malaise,  loss  of  appetite,  and  diarrhea  were  noted." 

As  was  previously  stated,  Noguchi  now  asserts  that  the  control 
fluid  may  be  omitted.  In  syphilitic  persons  this  fluid  may  give  a 
reaction  that  is  less  intense  and  that  is  not  followed  by  induration,  but 
is  due  probably  to  a  true  allergic  condition,  whereas  in  normal  persons 
none  or  but  a  slight  traumatic  reaction  occurs.  It  may  at  times  be 
difficult,  however,  to  distinguish  a  slight  luetin  reaction  from  a  well- 
marked  traumatic  reaction,  and  in  these  instances  an  opinion  may  be 
withheld  until  controlled  by  a  second  injection  in  the  other  arm  with 
control  fluid.  In  other  words,  while  the  control  fluid  need  not  be  used 
routinely,  it  is  well  to  have  it  at  hand  to  be  used  in  these  doubtful  cases. 

In  view  of  the  occasional  instances  in  which  the  reactions  are  re- 
tarded a  patient  should  be  observed  for  two  weeks  before  a  reaction  is 
regarded  as  negative. 

Results. — 1.  The  reports  of  Noguchi  and  of  a  number  of  different 
observers  show  that  the  luetin  reaction  is  generally  negative  in  the 
primary  and  secondary  (untreated)  stages  of  syphilis. 

2.  In  latent  and  tertiary  syphilis  Noguchi  has  reported  positive 
reactions  in  from  80  to  95  per  cent,  respectively,  and  the  reports  of 
others  have  showed  from  64  to  100  per  cent,  of  positive  reactions. 

3.  In  cerebrospinal  syphilis  positive  reactions  have  been  reported 
in  from  42  to  80  per  cent,  of  cases. 

4.  In  congenital  syphilis  the  results  have  varied  within  wide  limits — 
10  to  96  per  cent,  of  positive  reactions.     In  cases  under  one  year  of  age 
Noguchi  has  reported  about  23  per  cent.,  and  among  later  cases  96 
per  cent.,  of  positive  reactions. 

5.  While  a  few  observers  have  reported  positive  results  in  diseases 
other  than  syphilis,  it  is  frequently  very  difficult  absolutely  to  exclude 
syphilis,  and  the  general  consensus  of  opinion  is  unmistakably  to  the 
effect  that  in  this  country,  at  least,  the  luetin  reaction  is  specific  for 
syphilis.     Slight  reactions  may  be  obtained  in  frambesia  or  yaws  and 
leprosy. 

6.  Second  injections  of  luetin  apparently  do  not  give  positive  reac- 
tions in  non-syphilitic  cases. 

Practical  Value. — It  may  be  stated  in  general  that  the  chief  value  of 
the  luetin  reaction  is  in  the  diagnosis  of  those  occasional  cases  of  latent, 
tertiary,  or  congenital  syphilis  that  fail  to  react  positively  with  the 
Wassermann  reaction.  I  am  quite  convinced  that  in  the  majority  of 
cases  a  negative  Wassermann  reaction,  carefully  and  skilfully  per- 


MALLEIN    REACTION  647 

formed,  and  especially  with  an  antigen  reenforced  with  cholesterin,  is 
stronger  evidence  of  the  absence  of  syphilis  then  is  a  luetin  test.  On  the 
other  hand,  a  definitely  positive  luetin  reaction  may  be  regarded  as 
indicating  that  the  patient  is  or  has  been  syphilitic,  even  though  the 
Wassermann  reaction  is  negative. 

2.  Results  indicate  that  in  syphilis  the  luetin  reaction  persists  longer 
than  does  the  Wassermann  reaction.     This  is  to  be  expected  when  we 
assume  that  the  substance  in  the  blood-serum  of  a  syphilitic  responsi- 
ble for  the  Wassermann  reaction,  is  a  separate  reactionary  product  of 
the  body-cells  to  pallida  toxin,  the  allergic  luetin  reaction  being  due 
to  the  true  pallida  antibody.     The  toxin  is  believed  to  disappear  from 
the  body-fluids  within  a  few  weeks  after  all  the  spirochetes  have  been 
killed,  whereas  the  allergic  antibody  may  persist  for  longer  periods  of 
time,  as  it  does  in  other  conditions  and  diseases.     In  a  case  of  treated 
syphilis,  therefore,  showing  a  persistently  negative  Wassermann  reaction 
with  both  serum  and  spinal  fluid,  a  positive  luetin  reaction  does  not 
necessarily  indicate  that  further  treatment  is  required. 

3.  Briefly  stated,  to  determine  if  a  frankly  syphilitic  patient  requires 
further  treatment  the  Wassermann  reaction  with  serum  and  cerebro- 
spinal  fluid  is  the  better  test.     To  determine  more  definitely  whether 
a  given  person  showing  a  negative  Wassermann  reaction  has  ever  had 
syphilis  the  luetin  reaction  is  the  better  and  more  conclusive  test;    or 
if  in  such  a  person  the  Wassermann  reaction  is  positive,  the  luetin  test 
may  be  used  for  control  and  as  corroborative  evidence. 


MALLEIN  REACTION 

Mallein  is  a  glycerin  extract  containing  the  toxic  principles  of  the 
Bacillus  mallei,  the  microorganism  causing  glanders.  It  is  used  entirely 
as  a  diagnostic  agent  in  veterinary  practice,  but  may  also  be  used  for 
the  diagnosis  of  human  glanders,  the  dosage  being  the  same  as  that  of  old 
tuberculin. 

Two  methods  are  commonly  employed:  (1)  The  subcutaneous  in- 
jection and  (2)  instillation  into  the  eye  (ophthalmic  test). 

Method  of  Preparing  Mallein.1 — A  pure  culture  of  Bacillus  mallei  is  usually  ob- 
tained by  injecting  a  male  guinea-pig  with  infected  material,  and  at  the  end  of 
twenty-four  or  forty-eight  hours,  isolating  a  pure  culture  from  the  testicle. 

The  microorganism  is  grown  for  from  six  to  eight  weeks  in  special  bouillon 
containing  5  per  cent,  glycerin,  at  37°  C.,  similar  to  tuberculin.  Unlike  the  tubercle 

1  Technic  employed  by  the  Penna.  State  Live  Stock  and  Sanitary  Board,  Dr. 
A.  B.  Hardenburgh,  Director. 


648      ANAPHYLAXIS    IN    RELATION   TO    INFECTION   AND    IMMUNITY 

bacillus,  glanders  bacillus  grows  evenly  through  the  bouillon  instead  of  upon  the 
surface. 

At  the  end  of  six  or  eight  weeks  the  flasks  are  removed  from  the  incubator  and 
placed  in  a  sterilizer  at  100°  C.  for  at  least  two  hours.  This  process  kills  the  bacilli 
and  extracts  the  toxic  principles.  The  entire  solution  is  evaporated  down  to  TV  of 
its  volume,  filtered  in  small  bottles,  and  sterilized.  This  is  called  concentrated  mal- 
lein,  and  is  kept  in  this  form  until  ready  for  use. 

Before  using  it  is  diluted  to  its  original  volume  with  0.5  per  cent,  phenol  solution, 
and  passed  through  a  porcelain  filter.  This  process  removes  all  the  bacilli,  and  ren- 
ders the  solution  aseptic  and  ready  for  use. 

Ophthalmic  mallein  is  prepared  by  taking  one  part  of  concentrated 
mallein  and  adding  20  parts  of  absolute  alcohol.  This  forms  a  precipi- 
tate that  is  filtered  and  dried  in  a  desiccator  over  sulphuric  acid.  A 
5  per  cent,  solution  of  the  powder  is  made  with  sterile  water. 

Subcutaneous  Mallein  Reaction. — The  dose  of  mallein  for  a  horse 
is  0.4  c.c.  of  concentrated  mallein,  or  4  c.c.  or  1  dram  of  the  diluted 
product.  The  dose  for  a  retest  is  0.8  c.c.  of  concentrated  or  8  c.c.  or  2 
drams  of  the  diluted  solution.  Mallein  is  injected  subcutaneously  in 
some  convenient  area,  such  as  around  the  shoulder,  which  has  been 
shaved  and  cleaned. 

A  positive  reaction  is  based  on  the  same  principle  as  tuberculin, 
that  is,  a  rise  of  temperature  within  twenty-four  hours  following  the 
injection,  with  a  local  inflammatory  reaction  at  the  site  of  injection. 

Ophthalmic  Mallein  Reaction. — Two  or  three  drops  of  the  aqueous 
solution  are  dropped  into  the  inner  canthus  of  one  eye.  In  case  of  a 
positive  reaction  this  is  followed  by  a  marked  conjunctivitis,  associated 
with  a  purulent  exudate  extending  from  the  inner  canthus  similar  to  the 
reaction  shown  in  Fig.  128.  In  most  cases  there  is  also  a  rise  of  tempera- 
ture. Only  one  dose  is  to  be  applied  in  the  ophthalmic  mallein  test. 
It  is  not  considered  necessary  to  sensitize  the  eye,  as  in  tuberculosis. 
The  ophthalmic  mallein  test  has  the  same  advantages  as  the  ophthalmic 
tuberculin  test,  that  is,  one  can  obtain  a  reaction  when  dishonest  horse 
dealers  have  injected  mallein  prior  to  a  subcutaneous  mallein  test, 
also  in  cases  of  far-advanced  glanders,  which  at  times  give  no  reaction 
to  a  subcutaneous  injection  of  mallein. 

ABORTIN  REACTION 

Bang1  has  observed  that  artificially  infected  animals  reacted  with  a 
marked  rise  of  temperature,  loss  of  appetite,  and  slight  diarrhea  when  in- 
jected subcutaneously  with  a  culture  of  Bacillus  abortus,  which  he  had 

1  Ztsch.  f.  Tier.,  1897, 1,  241;  Archiv.  f.  Wissen.  und  Prakt.  Tierhl.,  1907,  33,  312. 


ALLERGIC    REACTIONS    IN    TYPHOID    FEVER  649 

established  as  the  course  of  infectious  abortion  of  cattle.  The  English 
Commission1  prepared  and  used  a  glycerine  extract  of  Bacillus  abortus 
in  the  same  manner  as  tuberculin,  and  with  success;  Meyer  and  Harden- 
bergh2  have  prepared  a  precipitated  purified  abortin  with  which  highly 
specific  reactions  have  been  observed;  Reichel  and  Harkins3  have  em- 
ployed an  intraderrnal  test  with  a  suspension  of  heat-killed  and  washed 
bacilli.  The  results  of  these  investigations  indicate  that  the  abortin 
test  is  highly  specific;  as  with  other  anaphylactic  reactions,  this  test 
does  not  serve  to  differentiate  the  actively  infected  from  the  recovered 
animals,  but  when  applied  to  a  herd  will  show  whether  or  not  Bang's 
disease  is  or  has  been  a  source  of  infection  among  the  animals. 

ALLERGIC  REACTIONS  IN  TYPHOID  FEVER 

In  1907  Chantemesse4  observed  characteristic  inflammatory  symp- 
toms follow  the  installation  of  typhoid  bacilli  extract  into  the  eye  of. 
patients  suffering  from  typhoid  fever.  Kraus 5  and  his  associates,  re- 
peating these  experiments,  could  not  convince  themselves  of  the  specific- 
ity of  this  reaction,  stating  that  healthy  individuals  also  give  it  to  some 
extent,  and  that  other  bacterial  extracts  cause  similar  symptoms  in 
typhoid-fever  patients.  In  addition  he  tried  a  cutaneous  reaction, 
but  without  result.  Zupnik,6  on  the  contrary,  states  that  a  cutaneous 
reaction  is  useful,  while  the  ophthalmic  reaction  is  not  useful.  Deehan 7 
obtained  a  weak  to  moderate  reaction  in  12  cases  of  typhoid  fever, 
whereas  eight  control  cases  showed  none.  Floyd  and  Barker  8  obtained 
positive  results  in  19  out  of  30  cases  and  none  in  18  controls,  including 
two  cases  of  paratyphoid  fever.  Chaufford  and  Trosier*  reported 
unfavorably  on  the  reaction. 

Austrian10  has  reported  very  favorably  upon  an  ophthalmic  reaction 
in  typhoid  fever  following  the  installation  of  "  typho-protein "  prepared 
by  cultivating  a  large  number  of  different  strains  of  typhoid  bacilli, 
precipitating  the  protein  with  alcohol,  drying  the  precipitate,  and  re- 

1  Report  of  Departmental  Committee  on  Epizootid  Abortion,  London,  1909, 
Part  I. 

2  Jour.  Infect.  Dis.,  1813,  13,  35 

3  Jour.  Amer.  Vet.  Med.  Assoc.,  March,  1917. 

4  Deut.  med.  Wchnschr.,  1907,  33,  1572. 

5  Wien.  klin.  Wchnschr.,  1907,  20,  1335. 

6  Munch,  med.  Wochenschr.,  1908,  45,  148. 

7  Univ.  Penn.  Med.  Bull.,  1909,  22,  6. 

8  Amer.  Jour.  Med.  Sci.,  1909,  38,  188. 

9  Compt.  rend.  Soc.  de'Biol.,  1909,  Ixvi,  519. 

10  Bull.  Johns  Hopkins  Hosp.,  1912,  23,  1. 


650      ANAPHYLAXIS    IN    RELATION    TO    INFECTION   AND    IMMUNITY 

dissolving  in  water  so  that  from  one-third  to  one-half  milligram  is 
contained  in  each  drop.  In  typhoid-fever  patients  reaction  of  the 
palpebral  conjunctiva  of  the  lower  lid  and  of  the  caruncle  appears  on 
an  average  of  two  and  a  half  hours  later,  reaching  the  maximum  about 
the  sixth  hour,  and  usually  subsiding  within  forty-eight  hours.  In  75 
cases  of  typhoid  fever  this  test  was  found  positive  in  71  and  negative 
in  four.  In  three  cases  the  eye  test  antedated  the  Widal  reaction,  and 
in  only  23  per  cent,  was  the  Widal  reaction  positive  at  as  early  a  date 
as  the  eye  test.  A  study  of  190  persons  normal  or  ill  with  diseases 
other  than  typhoid  has  convinced  Austrian  of  the  specificity  of  the  test, 
and  he  recommends  it  as  an  aid  to  diagnosis  on  account  of  its  simplicity 
and  the  absence  of  any  discomfort  to  the  patient. 

More  recently,  Gay  and  Force1  have  reported  favorably  upon  a 
cutaneous  reaction  indicative  of  immunity  against  typhoid  fever.  The 
preparation  which  they  used,  "typhoidin,"  is  prepared  in  the  same  man- 
ner as  Koch's  old  tuberculin:  250  c.c.  of  a  5  per  cent,  glycerin  bouillon 
is  inoculated  with  Bacillus  typhosus  and  cultivated  at  37°  C.  for  five 
days.  It  is  then  reduced,  without  filtration,  to  one-tenth  of  its  original 
volume  by  evaporation  over  an  acetone  bath  for  about  eight  hours. 
A  control  solution  of  sterile  5  per  cent,  glycerin  bouillon  is  prepared  in 
the  same  manner. 

The  skin  of  the  forearm  is  cleansed  with  alcohol,  and  two  abrasions 
are  made  with  the  von  Pirquet  borer,  as  described  under  the  cutaneous 
tuberculin  test.  The  "typhoidin"  is  applied  to  one  cut  and  the  control 
fluid  to  the  other.  The  reactions  are  observed  six  and  twenty-four 
hours  later.  Occasionally  there  is  a  traumatic  reaction  in  the  control, 
but  a  positive  reaction  may  be  detected  by  a  wider  areola  and  increased 
induration. 

Positive  reactions  were  secured  in  95  per  cent,  of  cases  that  had  re- 
covered from  typhoid  fever,  two  of  the  cases  having  had  the  disease 
respectively  forty-one  and  thirty-three  years  before.  The  reaction  was 
found  negative  in  85  per  cent,  of  individuals  not  having  typhoid  fever. 
Of  15  persons  immunized  by  the  army  method,  from  four  and  three- 
quarter  years  to  eight  months  previously,  nine  gave  a  positive  skin 
reaction.  Twenty-four  individuals  immunized  by  a  sensitized  vaccine 
(Gay  and  Claypoole)  for  from  one  to  eight  months  previously  reacted 
positively.  Later  Gay  and  Claypoole2  prepared  typhoidin  by  precipi- 
tating the  solution  with  alcohol,  washing  the  precipitate  with  alcohol 

1  Archiv.  Int.  Med.,  1914,  13,  471. 

2  Archiv.  Int.  Med.,  1914,  14,  671. 


ALLERGIC    REACTIONS    IN   TYPHOID    FEVER  651 

and  ether,  drying  in  a  vacuum,  and  suspending  the  resulting  powder  in 
phenolized  normal  salt  solution  which  was  injected  intracutaneously 
and  applied  cutaneously;  a  control  powder  was  prepared  from  broth 
and  used  in  the  same  manner.  With  this  skin  test  Gay  and  his  associates 
have  studied  the  relative  value  of  various  vaccines  and  regard  the  ana- 
phylactic  reaction  as  indicative  of  a  state  of  immunity.  Nichols1  has 
questioned  the  value  of  the  anaphylactic  skin  test  as  an  index  of  im- 
munity and  regards  the  reaction  as  indicating  nothing  more  than  sensi- 
tization  to  typhoid  protein,  which  is  apparently  less  lasting  and  less 
specific  than  the  true  immunity  to  this  infection.  He  bases  this  opinion 
on  the  fact  that  in  his  experience  the  typhoidin  skin  test  gave  fewer 
positive  reactions  (75  per  cent.)  than  generally  expected,  as  about  90  per 
cent,  of  persons  who  have  had  typhoid  fever  are  immune  for  many  years 
or  even  for  the  balance  of  life.  Furthermore,  according  to  Nichols, 
experience  has  shown  that  protection  following  typhoid  fever  is  of  longer 
duration  than  is  indicated  by  the  typhoidin  test,  and  while  a  large  per- 
centage of  persons  who  have  had  typhoid  fever  or  have  been  immunized 
with  typhoid  vaccine  react  to  paratyphoidin,  recent  experiences  and 
statistics,  particularly  in  Europe,  have  indicated  that  these  persons  are 
not  immune  to  paratyphoid  fever.  Kilgore2  has  reported  favorably  upon 
the  value  of  the  typhoidin  cutaneous  test;  Austrian  and  Bloomfield3 
found  that  the  test  failed  to  furnish  data  by  means  of  which  it  was  pos- 
sible to  differentiate  between  those  who  had  neither  typhoid  fever 
nor  had  received  the  vaccine  and  those  who  had  either  had  the  disease 
or  had  been  immunized. 

In  our  own  experience4  powdered  typhoidin  and  its  control  produced 
severe  reactions  when  injected  intracutaneously  in  doses  of  0.0005  to 
0.001  mgm.;  these  reactions  and  particularly  that  produced  by  the 
control  rendered  the  reading  and  interpretation  of  the  test  quite  difficult 
and  subject  to  much  error.  Cutaneous  anaphylaxis  to  typhoidin  was 
found  apparently  to  persist  for  a  longer  time  among  those  who  have  had 
typhoid  fever  than  among  those  actively  immunized  with  the  vaccine. 
Among  the  latter  the  highest  percentage  of  reactions  was  found  during 
the  first  year  following  immunization.  The  test  was  advocated  as  a 
means  of  determining  whether  or  not  a  person  possesses  immunity  to 
typhoid  fever  either  acquired  by  recovery  from  the  disease  or  by  arti- 

1  Jour.  Exper.  Med.,  1915,  22,  780. 

2  Archiv.  Int.  Med.,  1916,  17,  25. 

3  Archiv.  Int.  Med.,  1916,  17,  663. 

4  Jour.  Immunology,  1916,  1,  409. 


652      ANAPHYLAXIS   IN   RELATION   TO    INFECTION   AND    IMMUNITY 

ficial  immunization;  the  sum  total  of  researches  by  various  investigators 
is  to  the  effect  that  this  skin  test  is  an  indication  of  hypersensitiveness 
to  typhoid  protein,  but  cannot  be  accepted  at  present  as  an  indication 
of  immunity  (see  page  622) . 

ALLERGIC  REACTIONS  IN  OTHER  DISEASES 

Gonococcus  Infections. — In  1908  Irons1  reported  general  and  local 
reactions  in  persons  suffering  from  gonococcus  infections  following  the 
subcutaneous  injection  of  gonococcal  vaccines.  This  reaction  has  been 
observed  by  Brack2  in  epididymitis,  by  Reiter3  in  pelvic  infections  in 
women,  and  also  by  other  observers  in  other  conditions. 

Experiments  with  glycerin  extracts  of  the  gonococcus  prepared  from 
several  strains,  singly  or  combined  in  one  preparation,  have  yielded 
Irons4  well-defined  cutaneous  reactions.  These  tests  were  conducted 
after  the  method  of  von  Pirquet's  tuberculin  test. 

Diphtheria. — Shick  has  advocated  the  intracutaneous  injection  of  a 
minute  dose  of  diphtheria  toxin  as  a  test  for  antitoxin  in  the  serum  of  an 
individual.  If  sufficient  antitoxin  is  present,  the  toxin  is  neutralized 
and  no  local  disturbances  are  apparent;  otherwise  local  inflammatory 
areas  may  be  observed.  This  test  is  not  regarded  as  an  allergic  reac- 
tion; a  further  description  of  it  will  be  found  in  Chapter  XIV,  under 
Diphtheria  (see  page  228). 

Recently  Dr.  Moshage  and  I5  have  applied  an  anaphy lactic  skin  test 
in  diphtheria  with  a  polyvalent  antigen  designated  diphiherin;  posi- 
tive reactions  were  observed  in  about  70  per  cent,  of  children  and  35  per 
cent,  of  adults,  and  the  test  was  of  practical  interest  mainly  from  the 
viewpoint  that  the  anaphylactic  reaction  may  be  mistaken  for  a  positive 
Schick  reaction. 


ALLERGIC  REACTIONS  IN  SERUM  AND  FOOD  HYPERSENSITIVENESS 

Cutaneous  and  intracutaneous  reactions  have  been  utilized  in  the 
diagnosis  of  hypersensitiveness  to  horse  serum,  as  in  horse  asthma,  and 
to  the  proteins  of  various  foods,  as  egg-albumen,  milk,  shell-fish,  various 
vegetables,  etc. 

1  Jour.  Infect.  Dis.,  1908,  v,  279. 

2  Deut.  med.  Wchnschr.,  1909,  xxxv,  470. 

3  Zeitschr.  f.  Geburtsh.  u.  Kinderh.,  1911,  Ixviii,  471. 

4  Jour.  Amer.  Med.  Assoc.,  1912,  Iviii,  931. 
6  Amer.  Jour.  Dis.  Children,  1916,  xii,  316. 


ALLERGIC    REACTIONS    IN    SERUM    AND    FOOD    HYPERSENSITIVENESS    653 

A  cutaneous  test  for  serum  hypersensitiveness  is  described  on  page 
613  and  illustrated  in  Fig.  126.  An  intracutaneous  test  may  be  con- 
ducted by  injecting  into  the  skin  (not  subcutaneously)  0.1  c.c.  of  a  0.1 
per  cent,  sterile  solution  of  the  serum. 

Cutaneous  and  intracutaneous  tests  for  the  detection  of  food  idio- 
syncrasies are  described  on  page  616. 

Allergic  reactions  have  also  been  observed,  mostly  in  experimental 
animals,  in  leprosy,  sporotrichosis,  in  diseases  due  to  hyphomycetes,  and 
in  pregnancy.  Further  investigations  will,  no  doubt,  disclose  reactions 
of  practical  value  in  other  diseases,  such  as  rabies,  scarlatina,  measles, 
etc.,  following  the  successful  isolation  and  cultivation  of  their  respective 
causative  microparasites. 


CHAPTER  XXIX 

ACTIVE  IMMUNIZATION.— VACCINES  IN  THE  PROPHYLAXIS 
AND  TREATMENT  OF  DISEASE 

WHILE  the  importance  of  natural  immunity  must  not  be  under- 
rated in  the  protection  it  gives  us  after  bacterial  invasion  has  occurred, 
this  immunity  is,  however,  usually  relative  and  seldom  absolute,  and 
may  afford  insufficient  protection  if  the  invading  bacteria  are  numerous, 
or  particularly  virulent,  or  if  the  natural  resistance  of  the  organism 
is  weakened  by  fatigue,  disease,  or  injury. 

Usually  the  best  and  most  lasting  immunity  is  that  actively  ac- 
quired, in  which  our  own  body-cells  are  stimulated  or  trained,  as  it 
were,  to  produce  specific  antibodies  against  the  offensive  forces  of  a 
particular  bacterium  or  other  pathogenic  agent.  A  well-marked  and 
lasting  degree  of  this  form  of  immunity  usually  follows  recovery  from 
many  of  the  acute  infections,  particularly  the  acute  exanthemata,  such 
as  smallpox,  scarlatina,  measles,  typhoid  fever,  typhus  fever,  etc.  In 
other  infections,  such  as  erysipelas,  gonorrhea,  and  pneumonia,  the 
immunity  is  less  complete,  of  short  duration,  or  entirely  absent,  and, 
indeed,  a  state  of  hypersusceptibility  may  actually  follow. 

It  is  very  important,  in  this  connection,  to  remember  that  the  degree 
of  immunity  is  not  necessarily  in  proportion  to  the  severity  of  the  disease; 
thus  a  mild  infection  may  be  followed  by  the  much-desired  immunity, 
and  while  in  general  there  is  not  considerable  protection  without  infection, 
the  latter  does  not  necessarily  imply  that  a  virulent  infection,  or  even 
the  actual  disease  itself,  is  present,  for  discoveries  have  shown  that  an 
active  immunity  may  be  acquired  by  inoculation  with  the  antigen  so 
modified  or  attenuated  that  it  can  stimulate  the  production  of  specific 
antibodies  without  producing  the  disease  or  otherwise  greatly  disturbing 
the  health  of  the  individual. 

Historic. — The  facts  here  detailed  are  well  illustrated  in  the  history 
of  vaccination  in  smallpox  and  the  development  of  vaccine  therapy 
in  general.  Hundreds  of  years  ago  the  people  of  the  eastern  countries 
were  accustomed  to  expose  their  children  to  a  mild  case  of  smallpox  in 
order  that  a  similar  mild  infection  might  be  acquired  and  a  lasting  im- 

654 


HISTORIC  655 

munity  thus  secured  with  the  least  danger  to  life.  This  practice,  how- 
ever, was  not  without  risk,  as  the  mild  disease  not  infrequently  became 
a  virulent  one.  Later  the  dose  of  infectious  agent  was  decreased  by 
applying  the  virus  to  a  small  abrasion  on  the  skin,  and  the  resulting 
mild  but  genuine  attack  of  smallpox  usually  conferred  the  much-desired 
immunity.  But  here  again  the  severity  of  the  disease  was  not  under 
control,  as  the  virus  occasionally  assumed  increased  virulence  and  in- 
duced severer  infections  than  were  desirable.  Finally,  Edward  Jenner 
observed  and  showed  experimentally  (1796)  that  when  cowpox  virus  is 
inoculated  into  the  human  being,  a  trivial  infection,  since  called  "  vac- 
cinia," is  induced,  and  that  this  is  followed  by  an  absolute  or  nearly 
absolute  immunity  of  many  years'  duration  against  smallpox.  In  other 
words,  the  virus,  in  its  passage  through  the  cow,  becomes  so  modified 
that  it  can  no  longer  produce  smallpox,  but  is  still  able  to  stimulate 
the  production  of  the  specific  antibodies  against  this  disease.  Jenner 
worked  so  hard  to  establish  the  truth  of  this  finding  that  he  had  little 
time  to  devote  to  the  mechanism  involved  in  the  process. 

In  other  words,  the  work  of  Jenner  was  largely  empirical,  and  the 
explanation  was  not  forthcoming  until  many  years  later,  when  Pasteur 
laid  the  basis  of  scientific  immunization  by  discovering  that  light,  high 
and  low  temperature,  and  exposure  could  so  reduce  the  virulence  of  a 
microorganism  that  while  its  injection  into  an  animal  was  practically 
without  danger  or  ill  effect,  it  could  still  stimulate  the  protective  mech- 
anism of  the  host  and  induce  a  high  degree  of  immunity. 

That  this  could  be  done  was  a  fact  discovered  accidentally  by  Pasteur 
in  1879  while  working  with  the  organism  of  chicken  cholera.  After 
an  absence  from  home  he  found,  on  examining  his  cultures  on  his  return, 
that  they  had  become  innocuous — that  hens  could  bear  without  any  ill 
effect  inoculation  of  what  would  formerly  have  been  a  lethal  dose.  The 
prolonged  cultivation  of  the  microorganism  had  caused  its  attenuation 
and  Pasteur  immediately  grasped  the  far-reaching  importance  of  this 
discovery.  He  conjectured  that  it  might  be  possible  to  produce  a  mild 
and  modified  form  of  chicken  cholera  with  a  vaccine  of  the  attenuated 
microorganism  which  would  afford  protection  to  the  fowl  against  the 
severe  form  of  the  disease.  This  proved  to  be  the  case,  and  established 
the  possibility  of  so  modifying  or  attenuating  the  virulence  of  a  virus  or 
germ  that,  while  its  administration  is  not  followed  by  the  actual  disease, 
it  ts  capable  of  so  stimulating  the  body-cells  that  the  specific  antibodies 
are  produced.  This  discovery  formed  the  basis  of  prophylactic  im- 
munization or  bacterin  therapy  in  general. 


656 


ACTIVE    IMMUNIZATION 


Following  this  discovery,  much  work  was  done,  for  it  appeared  that 
the  question  of  prevention  of  any  bacterial  disease  simply  depended 
upon  whether  the  bacterium  could  be  cultivated  and  so  modified  or 
attenuated  that  while  its  injection  would  not  be  followed  by  disease  or 
other  harmful  effects,  it  would  be  capable  of  causing  the  production  of 
specific  antibodies. 

Naturally,  most  of  the  earlier  work  was  done  with  the  infections  of 
the  lower  animals,  and,  consequently,  most  discoveries  were  directly 
beneficial  to  them.  Pasteur  soon  devised  a  method  of  attenuating 
anthrax  bacilli  by  exposing  them  to  certain  temperatures  for  varying 
lengths  of  time,  so  that  a  vaccine  could  be  prepared  that  has  proved  of 
great  value.  Later  the  same  observer  discovered  a  method  of  attenuat- 
ing the  virus  of  hydrophobia  by  a  process  of  drying,  and  devised  a 
practical  method  of  prophylactic  immunization  against  this  disease. 
In  addition  to  these  his  vaccines  against  swine  erysipelas,  symptomatic 
anthrax,  and  rinderpest  have  become  well  known. 

The  knowledge  gained  from  a  study  of  the  diseases  of  the  lower  ani- 
mals and  the  aid  given  them  has  been  applied  to  human  medicine  with 
considerable  benefit,  not  only  in  prophylactic  immunization,  but  also 
in  therapeutics  (bacterin  therapy).  The  latter  application  is  a  more 
recent  discovery,  for  wnich  we  are  mainly  indebted  to  the  researches 
of  Wright,  Leishman,  Douglas,  and  their  colleagues. 

Nomenclature. — The  word  vaccine  is  from  the  Latin  vacca  (a  cow). 
Cowpox  was  called  "vaccinia,"  or  the  cow  disease,  and  Jenner  designated 
protective  inoculation  against  smallpox  with  cowpox  virus  as  vaccination. 
With  true  courtesy  Pasteur  adhered  to  Jenner's  nomenclature  and 
applied  the  term  vaccine  to  emulsions  of  dead  or  attenuated  bacteria. 
This  is  unfortunate  and  tends  to  create  confusion,  as  the  term  vaccine 
is  inseparably  associated  with  cowpox  virus  or  lymph.  The  term  bac- 
terial vaccine  has  become  widely  known,  and  is  used  to  designate  bacterial 
suspensions  prepared  for  purposes  of  immunization.  There  is  no  essen- 
tial difference,  however,  between  cowpox  vaccine,  which  contains  the 
modified  germ  or  virus  of  smallpox  in  a  diluent  of  lymph,  and  a  bacterial 
vaccine  containing  the  germ,  modified  by  some  physical  or  chemical 
agency  in  a  diluent  of  saline  solution  or  bouillon.  It  is,  however,  well 
to  reserve  the  unqualified  term  " vaccine"  for  cowpox  virus,  and  to 
retain  the  designation  "bacterial  vaccine"  for  suspensions  of  attenuated 
or  dead  bacteria.  More  recently  the  term  bacterin  has  been  applied 
to  the  latter,  but  this  would  imply  an  extract  of  bacteria,  as,  e.  g.,  tu- 
berculin, which  is  not  always  the  case. 


METHOD    OF    PREPARING    VACCINES  657 

Some  confusion  likewise  exists  as  regards  the  terms  serum  and  vaccine 
therapy.  Serum  therapy  is  a  process  of  passive  immunization  induced 
for  either  protective  or  curative  purposes  by  the  injection  of  the  blood- 
serum  of  another  animal  that  has  been  actively  immunized  by  inocula- 
tion with  bacterial  toxins  or  the  bacteria  themselves,  as,  for  instance, 
the  injection  of  diphtheria  or  tetanus  antitoxins.  Vaccine  or  bacterin 
therapy  is  a  process  of  active  immunization  brought  about  by  the  injec- 
tion of  the  bacteria  or  their  products  directly  into  a  patient.  Bacterial 
vaccines  that  are  simple  emulsions  of  dead  or  attenuated  bacteria 
are  not,  therefore,  serums,  and  the  indiscriminate  use  of  the  two  terms 
is  much  to  be  regretted. 

Method  of  Preparing  Vaccines. — It  may  be  stated  that,  in  general, 
the  specific  microorganism  or  virus  used  in  a  vaccine  should  be  modified 
as  little  as  possible,  or  just  sufficient  to  rob  it  of  its  disease-producing 
power.  For  example,  typhoid  bacterial  vaccine  is  prepared  by  suspend- 
ing the  bacilli  in  salt  solution  and  exposing  them  to  just  enough  heat 
to  modify  them  so  that  they  can  no  longer  multiply.  The  less  modifica- 
tion, the  better  the  vaccine.  If  the  exposure  is  too  prolonged  or  the 
temperature  too  high,  the  vaccinogenic  power  of  the  bacilli  is  destroyed, 
and  the  suspension  in  salt  solution  is  no  more  potent  or  of  no  greater 
value  than  the  salt  solution  itself.  Therefore  the  nearer  the  vaccine 
approaches  the  fully  viable  virus  or  microorganism,  the  more  potent  it 
will  be.  The  proper  preparation  of  a  vaccine,  therefore,  is  the  first 
step  to  successful  vaccine  therapy. 

Vaccination,  using  the  term  in  its  broadest  sense,  may  be  performed 
for  prophylactic  purposes  and  curative  immunization  in  the  following 
ways : 

1.  The  living  microorganism  may  be  inoculated.  This  is  the  ideal 
method,  but  for  obvious  reasons  has  not  been  generally  used  and  is  still 
in  the  experimental  stage.  It  is  based  upon  experimental  observations 
made  on  the  lower  animals  that  an  organism  may  be  so  introduced  as 
to  render  it  incapable  of  producing  disease,  but  may,  however,  stimulate 
the  production  of  specific  protective  antibodies.  Most  work  on  typhoid 
fever  is  at  present  being  done  in  the  Pasteur  Institute  at  Paris.  Evidence 
thus  far  indicates  quite  conclusively  that  the  typhoid  bacillus  is  unable 
to  produce  typhoid  fever  unless  it  is  introduced  into  the  gastro-intestinal 
tract,  and  the  subcutaneous  injection  of  living  bacilli,  modified  only  to  a 
slight  extent  by  artificial  cultivation,  is  not  followed  by  ill  effects  and 
produces  a  high  grade  of  immunity.  The  principle  is  a  good  one,  i.  e., 
in  a  vaccine  the  microorganism  should  be  modified  as  little  as  possible. 
42 


658  ACTIVE    IMMUNIZATION 

For  obvious  reasons  this  method  is  being  extensively  tried  out  on  the 
lower  animals,  especially  the  chimpanzee,  before  it  is  applied  to  human 
medicine. 

2.  By  inoculation  with  a  modified  virus  or  with  microorganisms  at- 
tenuated or  modified  according  to  various  methods. 

(a)  By  passing  the  virus  through  a  lower  animal,  as  the  passage  of 
smallpox  through  the  heifer  when  the  virus  is  incapable  of  producing 
smallpox,  although  vaccinia  confers  a  specific  immunity  against  small- 
pox. A  vaccine  for  swine  erysipelas  is  prepared  in  the  same  manner 
(Pasteur)  by  passing  the  bacillus  through  the  rabbit  several  times, 
which  increases  its  virulence  for  the  rabbit  but  decreases  it  for  swine. 

(6)  By  exposing  suspensions  of  microorganisms  to  heat.  They  are 
usually  grown  on  a  suitable  solid  medium,  suspended  in  salt  solution, 
and  exposed  to  a  temperature  at  or  just  above  their  thermal  death-point 
for  just  sufficient  time  to  kill  or  attenuate  them  in  so  far  that  they  can- 
not multiply.  The  same  result  may  be  secured  by  longer  exposure  to  a 
lower  temperature.  To  secure  a  potent  vaccine  the  principle  of  minimum 
exposure  at  the  minimum  temperature  should  be  observed,  the  question  of 
viability  being  controlled  by  culturing  the  vaccine.  Most  bacterial 
vaccines  are  prepared  in  this  manner.  Of  course,  the  fact  that  a  vaccine 
is  sterile  could  be  confirmed  by  exposing  it  to  a  very  high  temperature, 
but  in  this  case  the  product  may  be  of  no  more  value  than  so  much  salt 
solution.  Usually  an  exposure  of  53°  to  60°  C.  for  from  one-half  to  one 
hour  is  sufficient,  and  only  exceptionally  are  these  limits  exceeded. 

(c)  By  exposing  the  microorganism  to  air  and  light.     The  first 
bacterial  vaccine  (chicken  cholera)  was  accidentally  prepared  by  Pasteur 
in  this  manner. 

(d)  By  desiccating  or  drying  the  virus.     This  is  the  method  of 
vaccination  in  rabies,  as  the  virus  contained  in  the  spinal  cord  of  rabbits 
is  dried  for  varying  lengths  of  time,  emulsified,  and  injected.     The  longer 
the  period  of  drying,  the  greater  the  attenuation,  and  in  this  manner  the 
strength  of  the  vaccine  and  the  progress  of  immunization  are  under 
control. 

(e)  By  exposing  the  microorganism  to  a  high  temperature  for  vary- 
ing lengths  of  time.     Anthrax  vaccine,  for  the  immunization  of  lower 
animals,  is  prepared  in  several  strengths  by  exposing  suspensions  of  the 
bacilli  to  42°  C.  for  varying  periods  of  time. 

(/)  By  exposing  the  microorganisms  or  their  products  to  certain 
chemical  germicides,  as  in  the  preparation  of  anthrax  vaccine  by  Roux, 


MECHANISM    OF   ACTIVE    IMMUNIZATION  659 

diphtheria  and  tetanus  toxins  (Behring),  and  in  some  preparations  of 
tuberculin. 

3.  By  inoculating  with  bacterial  constituents,  as  the  soluble  toxins, 
bacterial  extracts,  and  products  of  bacterial  autolysis,  as  in  the  preparation 
of  Koch's  tuberculin  T.  R.,  Koch's  old  tuberculin,  mallein,  diphtheria 
and  tetanus  toxins,  etc. 

In  Chapter  XIII  a  method  is  given  for  preparing  bacterial  vaccines, 
of  which  typhoid  vaccine  is  a  type.  Special  methods  of  preparing  cer- 
tain bacterial  vaccines  and  other  vaccines,  such  as  cowpox  virus  and 
rabies  vaccine,  are  given  in  this  chapter. 

Mechanism  of  Active  Immunization. — As  was  stated  in  the  chapters 
on  Immunity,  in  the  presence  of  an  infection  the  host  endeavors  to  pro- 
tect itself  and  overcome  the  invaders  by  various  means,  among  which 
are  phagocytosis  and  the  production  of  more  or  less  specific  antibodies 
that  may  neutralize  the  poisons  of  the  parasite  (antitoxins),  directly 
kill  or  destroy  them  (bactericidans),  or  so  lower  their  vitality  or  re- 
sistance that  they  are  more  easily  phagocyted  (opsonins  or  bacterio- 
tropins). 

During  an  infection,  one  or  more  of  these  protective  forces,  or  all 
of  them,  are  brought  into  action.  After  the  infection  has  been  over- 
come, the  antibodies  do  not  always  disappear  at  once,  but  remain  for 
some  time  in  the  body-fluids  and  gradually  diminish,  so  that  if  the  host 
is  reinfected  with  the  same  parasite,  the  antibodies  are  at  hand  im- 
mediately to  overcome  it  and  protect  the  host  absolutely,  or  at  least 
so  to  modify  the  pathogenicity  of  the  parasite  or  neutralize  its  products 
that  the  host  will  suffer  but  mildly  while  the  parasite  is  being  finally 
destroyed.  The  concentration  and  duration  of  the  various  defensive 
forces  or  antibodies  vary  in  different  individuals  and  in  different  in- 
fections, so  that  the  degree  and  duration  of  an  active  acquired  immunity 
are  variable  factors.  Nevertheless — and  this  is  the  basis  of  active  im- 
munization— an  animal  or  a  person  may  have  specific  antibodies  for  a 
certain  parasite,  produced  by  its  own  cells,  without  actually  or  necessarily 
suffering  from  the  disease,  due  to  the  effects  of  inoculation  with  the 
germ  or  virus  in  a  modified  or  attenuated  form.  The  dose  of  vaccine 
may  be  so  controlled  that  general  symptoms  the  result  of  stimulation 
of  the  body-cells  are  slight  or  not  at  all  apparent,  and,  by  gradually 
increasing  the  dose,  more  and  more  antibodies  may  be  produced  until 
a  high  degree  of  immunity  is  secured. 

Active  immunization  may  be  practised  for  two  purposes: 

(a)  For  prophylaxis,  or  the  prevention  of  a  disease,  which  is  accom- 


660  ACTIVE    IMMUNIZATION 

plished  by  the  production  of  antibodies  so  that  they  may  be  at  hand  to 
overcome  an  infection  if  it  should  occur.  For  example,  the  antibodies 
specific  against  the  virus  of  smallpox  may  be  produced  by  inoculation 
with  cowpox  virus,  so  that  for  years  the  system  will  be  protected  against 
smallpox.  Even  if  vaccination  has  been  delayed  until  smallpox  has 
actually  been  contracted,  inoculation  with  cowpox  virus  early  in  the 
period  of  incubation  so  stimulates  the  body-cells  that  sufficient  anti- 
bodies are  produced  to  modify  and  lessen  considerably  the  virulence 
of  the  smallpox  virus. 

This  is  especially  true  in  rabies,  when  the  vaccine  is  given  in  such 
doses  and  at  such  intervals  that  sufficient  antibodies  are  produced  to 
neutralize  the  effects  of  rabic  virus  and  actually  to  destroy  it  during  the 
period  of  incubation  or  during  the  interval  that  elapses  between  the 
time  of  infection  and  the  appearance  of  the  symptoms.  In  this  way 
the  great  majority  of  infected  persons  escape  the  sufferings  of  rabies 
by  enduring  the  relatively  slight  discomfort  consequent  to  a  series  of 
subcutaneous  injections. 

(b)  For  the  treatment  of  disease.  This  is  the  bacterin  or  vaccine 
therapy,  a  method  that  owes  its  origin  to  the  researches  of  Sir  Almroth 
Wright  and  his  colleagues.  It  was  originally  employed  in  the  treatment 
of  those  infections  that  showed  a  tendency  to  chronicity  in  which  true  toxins 
played  little  or  no  part.  Since  recovery  from  an  infection  is  in  general 
dependent  upon  the  mechanical  removal  of  the  infecting  agent,  aided 
by  antibodies  that  facilitate  phagocytosis  or  directly  destroy  the  invading 
bacterium  and  neutralize  its  products,  Wright  believed  that  in  chronic 
infections  autovaccination,  or  stimulation  of  the  body-cells  to  the 
production  of  antibodies,  by  reason  of  the  fact  that  it  is  irregularly 
timed,  is  generally  insufficient  or  altogether  absent.  For  these  reasons 
he  believes  that  any  stimulus  that  will  arouse  the  body-cells  to  throwing 
into  the  circulation  substances  from  the  invading  bacterium  or  diseased 
tissues,  may  result  in  increased  antibody  formation,  followed  eventually 
by  clinical  improvement  or  cure.  In  certain  cases  this  stimulation  may 
be  secured  by  judicious  massage  or  manipulation  of  the  diseased  part, 
by  passive  hyperemia  (Bier),  or  by  similar  procedures. 

However,  if  the  microorganism  is  obtained  and  cultivated  artificially, 
it  is  possible,  in  many  instances,  so  to  modify  or  attenuate  the  bacilli 
(usually  by  heat)  that  they  may  be  reinjected  into  the  patient  in  suffi- 
cient numbers  to  furnish  the  stimulus  necessary  for  arousing  dormant 
or  uninvolved  body-cells  to  produce  the  opsonins  and  other  antibodies 
necessary  for  overcoming  the  infection.  In  other  words,  with  each  in- 


MECHANISM    OF   ACTIVE    IMMUNIZATION  661 

fection  the  host  endeavors  to  protect  itself  by  producing  antibodies. 
When  the  protection  is  insufficient,  the  infection  will  spread;  when  the 
antibodies  are  in  excess,  the  infection  is  overcome;  when  the  forces  are 
about  equal,  a  stage  of  chronicity  may  result  in  which  the  host  becomes 
accustomed,  as  it  were,  to  the  invaders,  and,  while  the  infection  does 
not  spread  rapidly,  it  does  not,  on  the  other  hand,  recede.  In  cases  of 
the  latter  type  an  extra  dose  of  bacterial  stimulant  (a  bacterin)  may 
arouse  dormant  or  inactive  cells  to  furnish  an  extra  quantity  of  anti- 
bodies and  thus  turn  the  tide.  In  acute  infections  indifferent  groups  of 
cells  may  likewise  be  brought  into  action,  although  it  is  more  likely  that 
they  are  already  involved,  so  that  the  extra  stimulation  in  the  form  of 
bacterin  must  be  cautiously  and  carefully  applied,  if  applied  at  all. 

In  therapeutic  inoculation,  therefore,  the  fundamental  principle  is  to 
stimulate  in  the  interest  of  the  infected  tissues  the  unexercised  immunizing 
capacities  of  the  uninfected  tissues.  This  is  especially  true  hi  chronic 
infections,  when  the  use  of  a  bacterial  vaccine  may  be  likened  to  the 
application  of  the  whip  to  a  lazy  horse  that  is  capable  of  further  effort 
and  work.  In  acute  infections,  however,  while  the  cells  are  at  work 
they  may  be  capable  of  greater  effort,  but  vaccines  should  be  given 
cautiously,  as  they  may,  to  use  the  same  simile,  act  as  a  whip  to  a  willing 
and  well-worked  horse  that  is  unable  to  respond  or  does  respond,  with 
resulting  disastrous  overexertion. 

It  should  be  remembered,  in  this  connection,  that  usual  forms  of  treat- 
ment should  be  given  while  bacterin  therapy  is  being  instituted.  For  in- 
stance, it  is  useless  to  administer  a  vaccine  to  a  patient  with  a  sup- 
purative  fistula  or  sinus  if  an  infected  silk  suture  is  directly  responsible 
for  the  suppuration.  The  suture  should  be  removed,  if  possible,  and 
after  this  is  done,  a  vaccine  may  be  of  considerable  aid  in  overcoming 
the  coincident  infection. 

In  what  manner  can  dead  bacteria  cause  the  production  of  anti- 
bodies? The  mechanism  is  similar  to  that  involved  during  infection 
with  the  living  microorganism,  and  involves  the  first  principles  of  im- 
munity. The  antigenic  powers  of  a  vaccine  are  probably  always  more 
or  less  inferior  to  the  living  antigen,  as  some  principle  may  be  lost  during 
heating,  drying,  passage  through  animals,  the  action  of  germicides,  etc. 
For  this  reason  living  vaccines  are  to  be  preferred,  although,  for  obvious 
reasons,  they  cannot  generally  be  employed  in  human  practice. 

Just  what  portion  of  the  bacterial  cells  is  mainly  antigenic  it  is 
difficult  to  determine,  for  it  probably  varies  with  different  species. 
With  a  true  toxin,  as,  for  example,  the  diphtheria  bacillus,  the  toxin 


662  ACTIVE    IMMUNIZATION 

constitutes  the  main  principle,  and  causes  the  production  of  an  anti- 
toxin as  its  main  antibody;  with  other  bacteria,  such  as  the  typhoid 
bacillus,  a  soluble  toxin  and  an  endotoxin  in  combination  with  the  protein 
of  the  bacterial  cell  are  probably  the  main  antigenic  factors  responsible 
for  the  formation  of  a  bacteriolysin,  opsonin,  antitoxin,  agglutinin,  etc. 

In  brief,  the  antigenic  principles  of  a  microorganism  are  mainly 
thermostabile  and  fairly  resistant  substances,  so  that  a  bacillus  may  be 
so  attenuated  or  altered  that  it  cannot  multiply  or  produce  disease,  and 
yet  is  capable,  through  the  agency  of  substances  that  have  escaped 
destruction,  of  causing  the  production  of  specific  antibodies. 

As  was  previously  stated,  vaccines  may  cause  the  production  of 
different  antibodies.  As  curative  agents,  however,  it  would  appear  that 
they  are  most  efficacious  in  those  infections  in  which  phagocytosis  is 
known  to  be  chiefly  concerned  in  the  defense  of  the  host,  e.  g.,  in  staphy- 
lococcus  infections.  As  shown  by  Wright  and  Douglas,  Neufeld  and 
Rimpau,  a  bacterial  vaccine  facilitates  phagocytosis,  not  so  much 
qualitatively  or  quantitatively  as  through  the  production  of  specific 
substances  that  act  directly  and  primarily  upon  the  bacteria  and  render 
them  more  vulnerable  to  phagocytosis  (opsonin  or  bacteriotropin). 
Wright  has  advised  a  method  of  opsonic  measurement,  previously 
described,  for  measuring  the  immunity  response,  but,  as  will  be  under- 
stood, while  the  opsonin  may  be  the  chief  antibody,  it  is  seldom  if 
ever  the  only  one,  so  that  the  opsonic  index  is  but  one  measure  of  de- 
fensive power. 

According  to  Vaughan,  a  microorganism  is  directly  responsible  for 
the  production  of  a  specific  proteolytic  ferment  capable  of  causing  the 
disintegration  or  destruction  of  the  bacterial  cell  and  its  products.  The 
ferment  is  the  antibody,  and  is  produced  during  an  infection  or  by  a 
vaccine  in  just  the  same  manner  as  antibodies  in  general  are  produced. 
In  other  words,  Vaughan  regards  antibodies  as  of  the  nature  of  pro- 
teolytic ferments;  thus  the  protein  of  the  microorganism  composing 
a  vaccine  produces  a  specific  proteolytic  ferment  capable  of  overcoming 
its  substratum  when  it  meets  the  latter  in  the  form  of  the  invading 
microorganism  of  an  infection. 

For  example,  the  tissues  affected  may  be  unable  to  produce  a  suffi- 
cient quantity  of  the  specific  ferment  necessary  to  overcome  the  infec- 
tion. The  injection  of  bacterial  protein  in  another  and  healthier  part 
of  the  body  leads  to  the  production,  in  this  locality,  of  a  specific  ferment 
that  is  conveyed  to  the  diseased  area  by  way  of  the  circulatory  system, 
and  aids  in  destroying  the  protein  of  the  infecting  microorganism  and  its 
tissues. 


THE    NON-SPECIFIC    ACTIVITY    OF    BACTERIAL    VACCINES  663 

The  Non-specific  Activity  of  Bacterial  Vaccines. — Owing  to  the 
weight  of  laboratory  investigations  and  the  fundamental  laws  of  speci- 
ficity in  immunity  reactions,  the  sole  efficacy  of  a  bacterial  vaccine  has 
been  generally  ascribed  to  the  production  and  activity  of  specific  anti- 
bodies, and  any  deviation  from  this  current  of  thought  has  been  received 
with  a  measure  of  skepticism  and  disapproval.  Scattered  throughout 
the  literature  are  the  reports  of  more  or  less  isolated  observations  that 
in  the  treatment  of  infections,  both  acute  and  chronic,  good  results  have 
followed  the  use  of  non-specific  substances.  The  subject  has  been  recent- 
ly studied  and  developed,  particularly  by  Jobling  and  Petersen.1  The 
favorable  results  first  reported  by  Krause,2  Ichikawa,3  and  others  by  the 
intravenous  injection  of  typhoid  vaccine  in  typhoid  fever  were  soon 
followed  by  the  further  reports  of  Kraus,  of  Luedke,4  and  of  Miller5  that 
equally  good  results  could  be  obtained  with  other  substances,  such  as 
colon  vaccine,  or  even  with  solutions  of  proteins,  albumose,  etc.,  in  this 
infection.  Mueller  and  Weiss,6  and  Saxe,  Bruck,  and  Kiralihyda7  have 
obtained  striking  results  in  arthritis,  especially  gonorrheal  arthritis, 
by  the  intragluteal  injection  of  sterile  milk  and  of  sodium  nucleinate, 
both  of  which  substances  cause  a  marked  reaction.  Smith8  calls  atten- 
tion to  the  value  of  an  anaphy lactic  reaction  in  gonococcus  infections 
obtained  with  horse  serum,  normal  or  antigonococcic,  it  matters  not, 
provided  the  allergic  phenomenon  is  manifested.  Miller  and  Lusk9 
report  striking  results  in  acute,  subacute,  and  chronic  arthritic  condi- 
tions of  various  types  by  intravenous  injections  of  typhoid  vaccines 
and  proteose.  It  is  probable  that  the  beneficial  results  occasionally 
reported  in  the  autoserum  treatment  of  various  skin  diseases  may  be 
due  to  the  same  non-specific  mechanism. 

In  the  mechanism  of  the  non-specific  activity  of  vaccines  Jobling 
and  Petersen  have  mentioned  (1)  the  probable  selective  stimulation  of 
the  hematopoietic  tissues  by  the  non-specific  stimulus,  resulting  in  the 
production  of  specific  antibodies;  (2)  the  production  of  hyperleukocy- 
tosis;  (3)  the  production  of  fever;  (4)  the  mobilization  of  proteolytic 
and  lipolytic  ferments,  and  (5)  an  increase  of  antiferment. 

Furthermore,  it  would  appear  that  in  many  cases  vaccines  have  a 

1  Jour.  Amer.  Med.  Assoc.,  1916,  66, 1753.       2  Wien.  klin.  Wchnschr.,  1915,  xx,  29. 
3  Ztsch.  f.  Immunitatsf.,  orig.,  1914,  xxiii,  32. 
4Miinch.  med.  Wchnschr.,  1915,  62,  321. 
6  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvi,  1756. 

6  Wien.  klin.  Wchnschr.,  1916,  xxii,  249. 

7  Munch,  med.  Wchnschr.,  1916,  Ixiii,  511. 

8  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvi,  1758. 

9  Jour.  An.er.  Med.  Assoc.,  1916,  Ixvii,  2016. 


664          ,  ACTIVE    IMMUNIZATION 

profound  and  favorable  influence  upon  metabolism,  increasing  elimina- 
tion and  tending  at  the  same  time  to  increase  body  weight.. 

It  is  apparent,  therefore,  that  the  administration  of  either  an  autog- 
enous or  stock  bacterial  vaccine  may  produce  beneficial  results  not 
only  through  the  production  of  specific  antibodies  but  also  by  means  of 
certain  general  and  non-specific  agencies.  To  secure  these  results  suffi- 
ciently large  doses  must  be  given.  It  is  well  known  that  we  possess 
general  defences  against  infection,  and  it  is  entirely  logical  to  assume 
that  these  are  operative  in  combating  disease  and  may  be  subjected  to 
stimulation  and  increased  activity.  In  my  opinion  we  should  still  prefer 
the  use  of  autogenous  vaccines,  increase  their  immunizing  powers  by 
more  attention  to  the  technic  of  their  preparation,  and  adhere  to  our 
belief  in  their  probable  specific  effects,  while  realizing  that  their  adminis- 
tration may  also  stimulate  non-specific  agencies  of  therapeutic  value. 
Further  investigations  are  required  to  produce  safe  and  satisfactory 
preparations  of  some  protein  adapted  for  this  form  of  therapy  if  the 
non-specific  activity  of  some  protein  is  alone  desired. 

In  this  form  of  therapy  with  non-specific  bacterial  vaccines,  typhoid 
vaccine  has  been  generally  administered  by  intravenous  injection.  Some 
reaction  is  apparently  necessary  for  therapeutic  results,  but  severe  and 
undesirable  reactions  may  be  produced  with  marked  rise  in  temperature, 
severe  headache,  and  chill.  If  this  form  of  therapy  is  employed  the 
physician  should  very  carefully  consider  the  possibility  of  these  severe 
reactions  following  an  injection  and  the  ability  of  the  patient  to  with- 
stand them.  Small  doses  of  typhoid  vaccine,  as  40,000,000  to  70,000,000 
bacilli,  should  be  selected  for  the  first  injection  if  given  intravenously, 
with  a  gradual  increase  until  some  fever  and  leukocytosis  result.  Subcu- 
taneous injections  with  larger  doses  may  be  tried,  as  they  are  safer  but 
slower  in  action.  The  usual  forms  of  therapy  and  particularly  the  re- 
moval or  treatment  of  foci  of  infection  should  receive  careful  attention. 
Until  this  form  of  treatment  is  better  understood  and  a  purified  protein 
provided  in  standardized  dosage^  the  physician  should  exercise  extreme 
caution,  and  particularly  with  intravenous  injections. 

Living  versus  Dead  Vaccines.— Barring  accidents,  the  employment 
of  a  living  virus  is  the  most  certain  way  of  calling  forth  a  maximum 
output  of  antibodies.  There  is  at  present  no  satisfactory  explanation 
for  this  except  that  heat-labile  substances  destroyed  in  the  ordinary 
preparation  of  bacterial  vaccines  have  antigenic  properties  (Smith). 
Living  vaccines  are  also  capable  of  penetrating  into  deeper  tissues, 
whereas  dead  vaccines  may  remain  where  they  are  deposited.  Similarly 
living  viruses  are  capable  of  exerting  a  continuous  action  and  of  de- 


AUTOGENOUS  VERSUS  STOCK  BACTERIAL  VACCINES       665 

livering  an  infinite  number  of  blows,  whereas  the  injection  of  a  dead 
virus  produces  an  interrupted  action  and  deals  but  a  single  blow.  The 
actual  dangers  of  using  a  living  vaccine,  as  the  possibility  of  it  being 
too  virulent  and  thus  producing  disease,  or  of  regaining  virulence  or 
producing  chronic  "carriers"  preclude  their  general  employment  in 
human  practice. 

Sensitized  Vaccines. — Besredka  and  Metchnikoff1  have  suggested 
a  plan  of  injecting  a  vaccine  composed  of  living  bacteria  that  have  been 
immersed  in  their  specific  immune  serum  or,  in  other  words,  have  been 
sensitized  (serobacterin).  They  believe  that  such  vaccines  produce 
practically  no  negative  phase,  but  only  slight  local  and  general  reaction, 
and  that  the  general  response  with  antibody  formation  is  facilitated. 
This  principle  is  supported  by  the  observations  of  Theobald  Smith, 
who  found  that  the  experimental  injection  of  a  toxin-antitoxin  mixture 
aids  the  dissemination  of  the  toxin  through  the  body  quite  generally, 
whereas  the  pure  toxin  is  chiefly  held  at  or  near  the  place  of  injection. 
This  diffusion  tends  to  cause  maximum  antibody  formation  over  an 
entire  portion  of  the  body  by  a  relatively  small  amount  of  free  or  easily 
dissociated  toxin  in  the  toxin-antitoxin  mixture.  Smith  inclines  to 
the  belief  that  a  similar  phenomenon  of  diffusion  may  occur  with  sensi- 
tized dead  bacteria. 

In  addition,  the  specific  immune  serum  may  aid  in  the  disintegration 
of  the  bacterial  cell,  either  through  the  attachment  of  a  bacteriolytic 
amboceptor  that  would  tend  to  lyse  the  bacterium  with  a  complement 
of  the  tissues,  or  through  a  preliminary  action  of  opsonin  which  prepared 
the  bacterium  for  ultimate  destruction  and  liberation  of  antigenic 
principles. 

Autogenous  versus  Stock  Bacterial  Vaccines. — It  may  be  stated  in 
general  that  autogenous  vaccines,  i.  e.,  those  prepared  from  the  pa- 
tient's own  bacteria,  should  be  used  whenever  possible,  especially  in 
the  vaccine  treatment  of  disease.  To  be  successful,  vaccine  therapy 
demands  that  the  bacteria  be  as  little  changed  as  possible.  Before 
they  are  killed,  the  bacteria  should  be  endowed  with  as  many  of  the 
potencies  as  possible  with  which  they  maintain  themselves  in  the  body. 
As  these  potencies  do  not  remain  unchanged  during  artificial  life,  as 
the  loss  of  capsules,  loss  of  virulence,  etc.,  it  is  advisable  to  secure  the 
organism  causing  the  infection  as  quickly  as  possible  and  prepare  a 
vaccine  without  undue  delay. 

Variants  may  occur  among  cultures  of  the  same  species,  and  the 
injection  of  one  strain  may  not  protect  against  another,  as  shown  by 
1  Ann.  de  Tlnst.  Pasteur,  1913,  xxvii,  597. 


666  ACTIVE    IMMUNIZATION 

Neufeld  for  the  pneumococcus.  In  the  use  of  an  autogenous  vaccine  this 
risk  of  using  an  alien  species  or  a  different  strain  is  reduced  to  a  minimum. 

In  some  cases  the  difficulty  of  securing  and  of  identifying  the  in- 
fective agent  may  be  so  great  that  much  time  is  lost  in  preparing  auto- 
genous vaccines,  as,  for  instance,  in  gonorrheal  and  tuberculous  in- 
fections, and  in  such  cases  it  may  be  necessary  to  use  a  stock  vaccine. 

In  protective  immunization  stock  vaccines  are  used,  as,  for  instance, 
in  the  preparation  of  typhoid  vaccine.  In  certain  instances,  as  in 
gonococcus  and  tuberculous  infections,  stock  vaccines  possess  but 
slightly  inferior  therapeutic  value  as  compared  with  autogenous  vaccines, 
not  to  mention  the  delay  and  difficulty  in  cultivating  and  preparing 
autogenous  vaccines.  In  many  other  infections,  as  with  the  Bacillus 
coli  and  gtreptococci,  stock  vaccines  possess  little  or  no  value. 

The  wholesale  manufacture  of  various  bacterial  vaccines  and  their 
indiscriminate  use  have  brought  disappointment  to  many.  Rational 
and  scientific  vaccine  therapy  does  not  consist  in  the  administration  of 
ready-made,  uncertain,  and  oftentimes  hit  or  miss  mixtures,  recently 
so  widely  exploited.  Especially  is  this  true  in  the  use  of  vaccines  for 
therapeutic  purposes.  The  bacteriotherapeutisjt  must  possess  sufficient 
skill  to  enable  him  to  make  bacteriologic  diagnoses,  prepare  autogenous 
vaccines,  and  skilfully  guard  their  administration.  In  most  instances 
these  requirements  are  fulfilled  by  cooperation  between  the  clinician 
and  bacteriologist,  or,  better,  by  one  who  is  especially  trained  in  vaccine 
therapy. 

The  Negative  Phase. — As  has  been  stated  in  a  previous  chapter, 
certain  local,  constitutional,  and  focal  disturbances  may  follow  the 
injection  of  a  bacterial  vaccine.  The  first  or  local  symptoms  are  not  in- 
frequently due  to  an  excess  of  preservative  in  the  fluid  or  to  the  presence 
of  contaminating  microorganisms,  but  abscess  formation  is  distinctly 
rare.  I,  in  common  with  others,  prefer  to  find  slight  focal  and  con- 
stitutional symptoms  following  the  first  one  or  two  doses  of  vaccine. 
In  furunculosis,  for  instance,  when  the  old  lesions  discharge  a  little  more 
and  one  or  two  others  threaten  to  develop  during  a  day  or  so  following 
the  injection  of  vaccine,  I  feel  assured  that  the  ultimate  result  will  be 
good.  These  reactions  are  particularly  desirable  for  the  non-specific 
effects  of  a  vaccine.  In  tuberculin  therapy,  however,  the  trend  of  opinion 
is  very  much  in  favor  of  administering  doses  so  small  that  no  appreciable 
focal  or  constitutional  lesions  will  follow.  Both  the  pathologic  a -id  the 
immunologic  process  concerned  in  tuberculosis  are  somewhat  different, 
and  in  ordinary  bacterin  therapy  a  slight  focal  disturbance  is  desirable 


CONTRAINDICATIONS   TO   ACTIVE    IMMUNIZATION  667 

and  indicates  that  the  vaccine  in  question  possesses  some  potency. 
Allen,  who  has  an  exceptionally  rich  experience  in  vaccine  therapy, 
frequently  mentions  the  desirability  of  administering  doses  sufficiently 
large  to  evoke  slight  reactions. 

Following  the  administration  of  a  vaccine  it  is  believed  that  the 
quantity  of  opsonin  in  the  body-fluids  is  temporarily  decreased,  and  that 
the  inoculated  person  is,  therefore,  more  susceptible  to  infection  (nega- 
tive phase)  (Fig.  56).  It  can  readily  be  understood  how  a  vaccine 
may  temporarily  depress  the  cells  and  defensive  mechanism  in  general, 
but  just  how  it  may  bring  about  an  actual  decrease  in  opsonin  it  is 
more  difficult  to  understand,  as  the  actual  amount  that  may  be  used 
in  dealing  with  the  vaccine  itself  must  be  small.  Veterinarians  are 
careful  not  to  expose  cattle  to  infection  immediately  after  they  are  im- 
munized with  anthrax  vaccine,  on  account  of  this  hypersusceptibility 
to  infection.  In  general,  however,  I  have  observed  that  most  im- 
munizators  are  prone  to  regard  the  question  lightly,  and  to  neglect 
the  importance  of  the  negative  phase,  whereas  some  deny  that  it  ever 
exists. 

Contraindications  to  Active  Immunization. — It  should  be  emphasized 
that  a  properly  prepared  vaccine  is  a  potent  substance  capable,  when 
given  in  excessive  dosage  or  when  otherwise  injudiciously  administered, 
of  doing  much  harm.  A  vaccine  stimulates  body-cells,  and  unless  the 
cell  can  withstand  the  stimulation,  the  administration  of  vaccine  may 
do  actual  harm.  This  is  the  main  reason  why  vaccines  should  be  used 
very  cautiously,  if  at  all,  in  the  treatment  of  severe  generalized  infections. 
In  passive  immunization  the  conditions  are  different,  as  the  body-cells 
are  not  taxed,  but  rather,  through  the  neutralization  of  the  toxic  sub- 
stances which  they  are  combating,  they  are  relieved,  and  an  antibody- 
laden  serum  may,  therefore,  be  freely  administered  in  severe  infections. 

In  active  immunization  for  therapeutic  purposes  the  conditions 
should  be  carefully  weighed  and  the  treatment  conducted  by  one  who  is 
qualified  to  judge  of  the  potencies  of  harm  and  good  in  a  vaccine,  and 
who  has  had  sufficient  experience  to  guide  him  in  dosage  and  frequency 
of  inoculation,  the  main  objects  being  to  tide  over  and  aid  nature  during 
an  acute  infection,  and  to  arouse  and  stimulate  her  during  a  chronic 
infection. 

In  prophylactic  immunization  the  physician  should  satisfy  himself 
that  the  patient  has  no  latent  or  active  infection  that  may  be  rendered 
worse  during  the  temporary  depression  that  follows  inoculation.  It  is 
true  that  this  depression  is  fleeting  and  temporary,  and  that  the  possi- 
ble harm  incurred  may  be  far  outweighed  by  the  ultimate  good,  but  vac- 


668  ACTIVE    IMMUNIZATION 

cines  should  be  given  with  proper  discernment  and  riot  carelessly  and 
injudiciously.  These  remarks  have  no  relation  to  cowpox  vaccination, 
where  the  good  so  far  overbalances  the  possible  harm  that  in  general 
all  persons  should  be  vaccinated,  especially  if  an  epidemic  is  impending. 

(a)  Tuberculosis. — There  is  at  present  some  discussion  relative  to 
the  harm  that  may  be  caused  in  tuberculosis  by  typhoid  immunization. 
Probably  all  will  agree  that  a  patient  with  an  active  and  acute  tubercu- 
lous lesion  should  be  refused  inoculation,  but  when  the  lesion  is  quiescent 
or  healed,  or  in  the  early  latent  stage,  it  is  indeed  difficult  to  understand 
how  a  prophylactic  dose  of  typhoid  vaccine  will  do  more  or  as  much 
harm  as  an  attack  of  tonsillitis,  rhinitis,  or  some  similar  acute  infection. 

(6)  In  diabetes,  carcinoma,  and  other  debilitating  conditions  vaccines 
should  not  be  administered  unless  the  indications  or  requirements  are 
unusually  urgent. 

(c)  Advanced  nephritis,  especially  parenchymatous  nephritis,  may 
be  regarded  as  contraindicating  the  administration  of  a  vaccine. 


PROPHYLACTIC  IMMUNIZATION  OR  VACCINATION 

SMALLPOX 

Historic. — Just  when  and  where  smallpox  vaccination  was  first 
practised  is  not  known.  The  original  method  of  inducing  immuniza- 
tion against  the  disease  by  introducing  the  virus  from  a  smallpox  pa- 
tient into  a  healthy  person  through  an  abrasion  of  the  skin  and  thus 
greatly  diminishing  the  virulence  of  the  disease  was  practised  by  the 
Turks  during  the  eighteenth  century,  the  chief  object  being  to  preserve 
the  beauty  of  the  young  Turkish  and  Circassian  women.  In  1878  Lady 
Mary  Montagu,  the  wife  of  the  British  Ambassador  at  the  Ottoman  court 
in  Constantinople,  observing  this  practice  among  the  Turks,  had  her 
own  son  and  daughter  inoculated  and  was  largely  instrumental  in  estab- 
lishing the  practice  in  Europe. 

As  regards  the  prophylactic  value  of  this  method  of  inoculation  in 
England  and  continental  Europe,  statistics  are  incomplete,  but  the 
literature  of  contemporary  writers  shows  that  protection  was  usually 
complete.  The  induced  disease  was  not,  however,  always  mild,  and  not 
infrequently  assumed  an  unexpected  virulence  that  not  only  proved  dis- 
tressing and  even  fatal  to  inoculated  individuals,  but  also  constituted  a 
source  of  infection  to  a  community.  While,  therefore,  the  underlying 
principles  were  sound,  and  while  these  early  attempts  at  preventive 
immunization  mark  an  epoch  in  the  history  of  medicine  and  of  the  world, 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  669 

it  was  not  until  Edward  Jenner  made  his  investigations  into  a  theory 
held  by  farmers  and  by  experimental  evidence  established  it  as  true  that 
a  satisfactory  method  of  immunizing  the  body  against  smallpox  was 
introduced. 

The  peasantry  in  various  parts  of  Europe,  and  especially  in  England, 
had  generally  observed  that  those  who  had  had  sores  on  their  hands  con- 
tracted from  similar  lesions  on  the  teats  of  cows,  usually  escaped  small- 
pox infection  when  the  disease  was  epidemic  in  a  community.  In  fact, 
it  is  said  that  several  farmers  deliberately  inoculated  members  of  their 
family  with  cowpox  lesions  and  that  these  escaped  smallpox. 

Edward  Jenner  was  a  physician  practising  in  Berkeley,  Gloucester- 
shire, and  frequently  used  the  method  of  direct  inoculation  from  a  mild 
case  of  smallpox  among  his  patients.  While  a  student  he  was  impressed 
with  the  traditions  of  cowpox  vaccination,  and  finding  that  they  were 
largely  true,  determined  to  make  experimental  tests.  On  May  14, 
1796,  he  vaccinated  a  boy,  James  Phipps,  with  virus  from  a  cowpox 
lesion  on  the  hand  of  a  dairy  maid,  Sarah  Nehnes,  and  on  July  1st  he 
inoculated  the  same  boy  with  pus  from  a  smallpox  patient  without  re- 
sulting infection.  In  1798  he  furnished  further  proof  that  cowpox  will 
afford  protection  against  smallpox  by  inoculating  a  child  direct  from  a 
vesicle  on  the  teat  of  a  cow,  and  continued  the  inoculation  from  arm  to 
arm  through  a  series  of  five  children,  after  which  all  were  inoculated 
with  smallpox  virus,  without  a  single  case  developing.  In  the  same  year 
he  published  "An  Inquiry  into  the  Causes  and  Effects  of  the  Variolse 
Vaccine/'  illustrated  by  four  plates,  and  within  a  year  or  two  vaccina- 
tion became  general  over  Europe. 

Vaccination  was  introduced  into  the  United  States  in  July,  1800,  by 
Dr.  Benjamin  Waterhouse,  Professor  of  Physic  at  Harvard  University, 
who  vaccinated  his  own  children.  At  about  the  same  time  John  Red- 
man Coxe,  of  Philadelphia,  vaccinated  his  oldest  child  and  then  tested 
the  experiment  by  exposing  him  to  cases  of  smallpox.  This  bold  repeti- 
tion of  Jenner's  experiment  considerably  strengthened  public  confidence 
in  the  method  and  the  practice  spread  rapidly.  Thomas  Jefferson,  writ- 
ing in  1806  to  Edward  Jenner,  said:  "Future  generations  will  know  by 
history  only  that  the  loathsome  smallpox  existed  and  by  you  has  been 
extirpated." 

But  Jenner  and  his  earlier  supporters  met  with  much  opposition, 
often  bitter  and  unrelenting,  and  this  is  readily  understood  when  it  is 
realized  that  even  at  the  present  day,  over  a  hundred  years  later,  cow- 
pox  vaccination  still  has  its  opponents,  in  spite  of  the  fact  that  the 


670  ACTIVE    IMMUNIZATION 

value  of  the  method  has  been  established,  and  it  has  been  found  the 
greatest  of  all  boons  to  the  human  race,  and  notwithstanding  that  it 
has  been  definitely  proved  that  a  thorough  and  continuous  practice 
of  the  operation  would  quickly  eradicate  smallpox  from  the  face  of  the 
earth.  This  opposition  is  especially  pernicious  and  unjust,  since  the 
practice  of  former  years  of  vaccinating  by  direct  transmission  from  arm 
to  arm  has  been  entirely  abandoned,  and  that  animal  lymph,  prepared 
and  collected  under  strict  aseptic  precautions,  is  being  used  exclusively. 

The  Relationship  of  Variola  and  Vaccinia. — The  relationship  of 
variola  to  vaccinia  has  been  discussed  since  Jenner's  time,  but  no  ade- 
quate explanation  has  been  found. 

According  to  the  general  belief  the  smallpox  virus,  whatever  it  may 
be,  is  altered  in  its  passage  through  a  lower  animal,  and  loses  forever  its 
power  of  producing  smallpox,  but  is  still  so  closely  related  that  the  anti- 
bodies it  produces  are  sufficient  to  protect  against  smallpox. 

The  close  interrelationship  existing  between  vaccinia  and  variola  is 
shown  by  the  presence  in  the  virus  of  both,  and  in  section  of  the  skin  of 
both,  of  microscopic  cell  inclusions,  first  described  by  Guarinieri  in  1892. 
This  finding  has  been  confirmed  by  Pfeiffer  in  Germany  and  Councilman 
and  his  associates  in  this  country.  These  investigators  have  made 
extensive  studies  of  these  bodies,  and  believe  them  to  be  protozoa  in- 
timately associated  with  the  etiology  of  vaccinia  and  variola.  More 
recently,  Fornet  has  described  certain  small,  diplococcus-like  bodies 
that  were  found  in  cowpox  vaccine  and  in  smallpox  lesions.  These  are 
regarded  as  having  an  etiologic  relationship  to  smallpox,  and  if  these 
findings  are  confirmed,  would  prove  the  identity  of  variola  and  vaccinia. 

Recent  investigators,  particularly  Copeman,  erf  England,  and  Brink- 
erhoff  and  Tyzzer,  of  America,  have  shown,  by  carefully  conducted  ex- 
periments, that  vaccination  will  protect  monkeys  against  subsequent 
inoculation  with  smallpox  virus,  and  this  completely  confirms  the  early 
experiments  of  Jenner  and  others  who  proved  the  efficacy  of  vaccination 
by  the  "variolous  test." 

The  Preparation  of  Cowpox  Vaccine. — During  the  early  days  of 
vaccination  it  was  customary  to  inoculate  human  beings  with  material 
obtained  from  the  pustules  of  those  previously  vaccinated.  The  old- 
time  physician  carefully  removed  choice  scabs  and  carried  them  about 
in  a  special  case  ready  for  inoculation.  While  this  method  served  its 
purpose  well,  there  were  several  drawbacks  to  its  use,  the  chief  of  which 
was  the  danger  of  transmitting  syphilis.  It  has  now  for  many  years 
been  the  custom  to  use  virus  obtained  from  animals,  the  production  of 


PROPHYLACTIC    IMMUNIZATION    OR   VACCINATION  671 

which  can  be  carefully  controlled  and  tested,  any  danger  of  transmitting 
syphilis  being  thus  obviated,  because  the  heifer  or  cow  used  in  the  pre- 
paration of  the  virus  is  not  subject  to  this  disease.  The  opponents  to 
vaccination,  however,  persist  in  using  old  and  obsolete  statistics  regard- 
ing the  transmission  of  syphilis  to  support  their  claims,  although  these 
have  absolutely  no  bearing  upon  the  modern  methods  of  preparing  the 
virus. 

Seed  Virus. — This  refers  to  the  virus  for  vaccinating  the  calves  or 
other  animals  used,  and  is  a  most  troublesome  factor  to  those  engaged 
in  this  work.  According  to  Park  and  Huddleston,  a  sufficient  amount  of 
vaccine  virus  should  be  on  hand  to  vaccinate  from  40  to  50  persons. 
Five  children  in  good  health  and  not  previously  vaccinated  should  then 
receive  an  inoculation,  each  spot  being  of  the  size  of  a  ten-cent  piece. 
On  the  fifth  day  after  vaccination  the  upper  layer  of  the  resulting  vesicle 
should  be  removed,  and  sterilized  bone  slips  be  rubbed  on  the  base  thus 
exposed.  From  100  to  200  slips  on  each  side  of  the  slip  may  be  charged 
from  each  child.  The  slips  should  be  allowed  to  dry  for  a  minute,  and 
should  then  be  placed  in  a  sterilized  box  and  preserved  in  cold  storage, 
where  they  will  remain  active  for  at  least  two  or  three  weeks.  The 
aforenamed  observers  now  use  rabbits  alternately  to  obtain  seed  virus. 

Subsequent  animals  are  vaccinated  with  any  one  of  three  vaccines — 
(1)  Slips  charged  from  typical  vesicles  of  a  calf;  (2)  slips  charged  with 
the  serum  from  a  calf  after  removal  of  the  vesicles;  (3)  the  glycerinated 
virus  may  be  used  to  vaccinate  succeeding  calves,  but  in  this  case  it  is 
necessary  to  keep  the  glycerinated  virus  for  two  or  three  months,  since 
the  use  of  fresh  virus  on  a  succession  of  calves  leads  to  prompt  degenera- 
tion of  the  vaccine  and  to  the  production  of  infected  vesicles. 

The  New  York  Vaccine  Laboratory  produces  a  virus  that  is  never 
more  than  four  successive  transfers  from  a  human  case  of  vaccinia, 
and  is  guaranteed  to  give  100  per  cent,  of  "takes"  in  primary  vaccina- 
tion. 

Animals. — Various  animals  have  been  used,  but  female  calves  from 
two  to  four  months  of  age  are  preferable.  Older  animals  may  be  used, 
and  in  several  European  institutes  cows  are  usually  employed.  With 
properly  constructed  operating-tables,  they  may  be  handled  with 
comparative  ease.  Rabbits  have  also  been  used,  especially  in  pro- 
pagating the  seed  virus  and  to  obtain  pure  and  highly  active  viruses. 

The  calves  are  kept  under  supervision  for  at  least  a  few  days.  In 
some  institutes  they  are  tested  with  tuberculin,  although  with  good 
veterinary  inspection  this  test  is  not  necessary.  Soon  after  admission 


672  ACTIVE    IMMUNIZATION 

the  animal  is  clipped  and  given  a  thorough  cleansing,  which  includes  the 
feet  and  the  tail. 

On  the  day  before  vaccination  is  to  be  performed  the  belly-wall  is 
cleanly  shaved  from  the  cuneiform  cartilage  to  the  pubis,  and  well  up 
on  the  inner  sides  of  the  thighs  and  the  flanks.  The  skin  is  then 
thoroughly  washed.  Just  preceding  the  vaccination  the  animal  is 
fastened  to  the  operating-table  and  the  abdomen  and  inner  surface  of 
the  thighs  prepared  as  for  an  aseptic  abdominal  section,  i.  e.,  a  thorough 
scrubbing  with  hot  water,  green  soap,  and  soft  brush,  followed  by  alcohol 
and  sterilized  water,  the  parts  being  then  dried  with  a  sterile  towel. 
All  other  parts  are  covered  with  sterile  sheets,  and  the  calf  is  now  vac- 
cinated under  aseptic  precautions. 

Vaccination. — About  100  small  scarifications  are  now  made  in  these 
areas,  preferably  by  cross-scratches  or  in  rows  of  lines  about  one  to 
two  centimeters  square  and  at  least  one  to  two  centimeters  apart. 
The  scarification  is  simple,  but  usually  brings  a  small  amount  of  blood. 
After  they  have  been  made,  they  are  mopped  with  sterile  gauze  and 
rubbed  with  the  charged  slips,  using  one  or  two  slips  for  each  small  area, 
depending  on  the  amount  of  virus  each  slip  contains.  The  lesions  are 
allowed  to  dry,  and  are  then  covered  with  sterile  gauze  or  a  simple 
protective  paste,  or  are  left  entirely  uncovered. 

Precautions  should  be  taken  to  keep  the  animals  as  clean  as  possible. 
Inoculated  animals  are  to  be  kept  in  stalls  or  stables  apart  from  those 
under  observation.  The  stable  should  be  so  constructed  that  the  floors 
can  be  flushed  daily  with  a  hose  and  hot  water.  Excreta  should  be  re- 
moved promptly.  No  bedding  is  permissible,  and  means  should  be 
provided  for  fastening  the  legs  and  preventing  the  animal  from  kicking 
the  scarifications. 

Collection. — Ordinarily,  within  forty-eight  hours  of  vaccination, 
the  scratches  are  pinkish,  slightly  raised,  and  papular,  and  within  five 
or  six  days,  depending  upon  the  rate  of  development  of  the  vaccine 
vesicles,  the  virus  should  be  ready  for  collection  (Fig.  131).  The  calf 
is  killed  and  placed  upon  the  operating-table.  The  appointments  of  the 
operating-room  are  usually  equal  to  those  in  a  well-equipped  hospital 
operating-room,  being  supplied  with  all  conveniences  and  means  for 
carrying  out  a  careful,  painstaking,  and  aseptic  technic. 

The  exposed  parts  are  covered  with  sterile  sheets.  The  operator 
and  his  assistant  are  clad  in  aseptic  gowns.  The  vaccinated  field  is 
thoroughly  scrubbed  with  soap,  sterile  water,  and  gauze,  and  mopped 
with  sterile  gauze.  Crusts  are  carefully  picked  off,  and  the  soft,  pulpy 


PROPHYLACTIC    IMMUNIZATION    OR   VACCINATION  673 

mass  cureted  off  with  a  special,  spoon-like  curet  and  collected  in  a 
sterile  vessel.  After  the  curetage,  serum  exudes  from  the  excoriated 
base  of  the  vesicle,  and  ivory  tips  may  be  charged  in  this.  The  sticky 
and  pulpy  exudate  is  then  mixed  with  four  times  its  weight  of  glycerin 
and  water  (50  per  cent,  glycerin,  49  per  cent,  water,  1  per  cent,  phenol), 
and  this  is  done  most  effectively  by  passing  the  mixture  between  the 


I 


FIG.  131. — PRODUCTION  OF  COWPOX  VACCINE. 

Note  the  lines  of  cowpox  lesions  over  the  abdomen  and  flanks  of  the  calf.  The 
surgeon  is  about  to  cleanse  this  area  in  a  thorough  and  careful  manner,  after  which 
the  cowpox  material  is  removed  with  a  curet  and  collected  in  a  sterile  vessel.  All 
precautions  are  taken  to  insure  as  thorough  aseptic  technic  as  possible. 

rollers  of  a  Doring  mill.  The  glycerinated  pulp  is  allowed  to  stand  for 
three  or  four  weeks  in  order  to  allow  bacteria,  which  are  invariably 
present,  to  undergo  dissolution.  At  the  end  of  this  time  the  glycerinated 
pulp  is  thoroughly  titrated  in  specially  constructed  triturating  machines, 
and  put  up  in  small  capillary  tubes,  which  are  sealed,  or  "vaccine 
points"  may  be  prepared.  If  properly  preserved  in  sealed  tubes  in 
a  dark,  cool  place  the  virus  should  remain  active  for  at  least  three  months. 
43 


674  ACTIVE    IMMUNIZATION 

According  to  Park  and  Huddleson,  10  grams  of  pulp  and  200  charged 
slips  would  be  an  average  yield  from  a  calf,  and  when  made  up  should 
suffice  to  vaccinate  at  least  1500  persons.  Calves  vary  greatly  in  their 
yield  of  virus.  Of  two  calves  vaccinated  in  exactly  the  same  manner, 
one  may  furnish  material  for  500  vaccinations  and  the  other  for  10,000 
inoculations. 

Testing  the  Vaccine. — The  virus  may  be  tested  for  its  efficacy  by  a 
variety  of  methods.  Calmette  and  Guerin 1  inoculate  rabbits  upon  the 
inner  surfaces  of  the  ears  and  estimate  the  potency  of  the  virus  from  the 
speed  of  development  and  the  size  of  the  resulting  lesions.  Guerin2 
estimates  the  potency  of  virus  quantitatively  by  inoculating  rabbits 
with  serial  dilutions  ranging  from  1  :  10  to  1  :  100.  Fully  potent  virus 
should  cause  closely  approximated  vesicles  in  a  dilution  of  1  :  500,  and 
numerous  isolated  vesicles  in  a  dilution  as  high  as  1  :  1000. 

Quantitative  estimation  of  the  bacteria  in  the  glycerinated  virus 
is  made  by  the  plating  method,  and  the  vaccine  used  only  when  the 
numbers  of  bacteria  have  been  greatly  diminished  or  are  entirely  absent. 
The  vaccine  is  also  tested  for  tetanus  by  injecting  relatively  large 
quantities  subcutaneously  into  guinea-pigs  and  mice. 

Under  the  Federal  Law  of  July  1,  1902,  and  the  regulations  framed 
thereunder,  all  firms  manufacturing  vaccine  virus  are  required  by  the 
Secretary  of  the  Treasury  to  obtain  a  license  before  they  may  sell  their 
products  in  interstate  commerce.  The  vaccine  laboratories  are  care- 
fully inspected  by  an  official  of  the  Hygienic  Laboratory  of  the  United 
States  Public  Health  and  Marine-Hospital  Service;  the  inspector 
carries  away  with  him  as  many  samples  of  virus  as  he  wishes,  and  addi- 
tional samples  are  purchased  in  the  open  market  in  different  parts  of  the 
country.  All  these  are  subjected  to  a  vigorous  bacteriologic  examina- 
tion, especially  for  tetanus  bacilli,  by  a  laboratory  worker  who  devotes 
all  his  time  to  this  work.  The  federal  regulations  require  each  vaccine 
institute  to  perform  a  careful  autopsy  on  each  calf  after  the  vaccine 
virus  has  been  removed,  and  if  any  communicable  disease  is  found  or 
suspected  in  the  animal,  the  virus  must  not  be  placed  on  the  market, 
but  must  be  destroyed.  In  accordance  with  this  law,  permanent  records 
of  the  bacteriologic  examinations  of  the  virus  and  of  the  autopsy  shall 
be  kept  in  each  institute. 

Noguchi's  Method  of  Preparing  Cowpox  Virus. — Noguchi3  has  suc- 
ceeded in  freeing  vaccine  virus  from  all  associated  bacteria  by  means  of 
1  Ann.  de  1'Inst.  Pasteur,  1902.  2  Ann.  de  1'Inst.  Pasteur,  1905. 

8  Jour.  Exper.  Med.,  1915,  21,  539. 


PROPHYLACTIC    IMMUNIZATION   OR   VACCINATION  675 

suitable  disinfecting  agents,  and  propagated  the  pure  virus  in  the  tes- 
ticles of  rabbits  and  bulls.  The  virus  cultivated  in  this  manner  is  not 
only  devoid  of  bacteria,  but  appears  capable  of  definite  transfer  from  one 
animal  to  another.  The  multiplication  of  the  virus  within  the  testicle 
was  found  by  Noguchi  to  reach  a  maximum  on  the  fourth  or  fifth  day 
after  inoculation,  with  diminution  after  the  eighth  day.  Skin  lesions 
produced  in  rabbits  and  calves  with  the  original  and  purified  or  bac- 
teria-free viruses  were  found  identical,  and  persons  have  reacted  to  the 
latter  in  an  entirely  typical  manner.  These  results  are  of  great  value 
and  importance  and  tend  to  increase  the  safety  of  vaccination  and  inci- 
dently  weaken  the  arguments  and  contentions  of  antivaccinationists. 

Technic  of  Vaccination. — The  essential  part  of  the  process  of  vac- 
cination is  that  the  virus  should  be  introduced  through  the  epidermis 
so  as  to  be  absorbed  by  the  lymphatics  and  blood-vessels  of  the  corium. 

The  site  usually  chosen  is  the  skin  of  the  outer  side  of  the  upper  arm, 
over  the  insertion  of  the  tendon  of  the  deltoid  muscle.  Sometimes,  in 
females,  the  outer  side  of  the  thigh  or  well  above  the  knee  on  the  inner 
aspect  of  the  thigh  is  used.  Vaccination  on  the  leg,  however,  is  never 
advisable,  as  it  would  appear  that  such  vaccinations  are  more  prone 
to  take  on  an  excessive  inflammatory  action  owing  to  the  greater  con- 
gestion due  to  the  dependent  position  of  the  lower  extremities;  there 
is  also  more  likelihood  of  secondary  infection  and  mechanical  violence 
occurring.  • 

The  skin  is  washed  with  alcohol  and  finally  with  water  and  then 
dried,  care  being  exercised  not  to  rub  too  vigorously;  if  the  skin  is 
reddened,  it  is  best  to  wait  until  the  hyperemia  subsides.  Grasp  the 
arm  with  the  left  hand,  rendering  the  skin  tense,  and  with  a  sterile 
needle  or  scalpel  carefully  remove  the  epidermis  over  a  square  area 
measuring  about  one-eighth  of  an  inch  (Fig.  132).  Bleeding  should  be 
avoided — an  abraded  surface  that  just  oozes  serum  is  especially  to  be 
desired.  The  virus  is  then  expressed  or  placed  upon  this  area  (never 
blown  out),  and  thoroughly  rubbed  in  with  a  sterilized  wooden  tooth-pick 
or  the  vaccine  point.  After  allowing  the  lymph  to  dry,  a  light  sterile 
gauze  dressing  should  be  applied. 

Some  operators  abrade  through  the  lymph;  others  make  one  or  two 
straight  lines  of  scratches  and  rub  in  the  virus. 

Another  excellent  method1  consists  in  abrading  the  arm  with  a  light 

rotatory  motion  of  a  von  Pirquet  chisel  (sterilized  in  alcohol  and  in  a 

flame),  measuring  about  2  mm.  in  width.     (See  Fig.  126.)     The  virus 

is  then  applied,  and  thoroughly  rubbed  in  with  a  sterile  tooth-pick. 

1  Force,  J.  N. :  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1466. 


676 


ACTIVE    IMMUNIZATION 


Cross-scarification,  which  is  forbidden  in  Germany,  favors  the  growth  of 
anaerobic  bacteria  under  the  crust  that  forms  on  the  surface  of  the 
abrasion  where  the  resistance  is  lowered  by  the  action  of  the  virus. 
The  circular  scarification  gives  more  control  over  the  dosage,  and  there 
is  no  tendency  to  the  development  of  excessively  sore  areas. 


FIG.  132. — METHOD  OF  VACCINATION  AGAINST  SMALLPOX. 

The  skin  is  stretched,  and  a  series  of  superficial  and  parallel  scratches  made  through 
the  epidermis  with  a  sterilized  needle. 

Usually  one  inoculation  of  the  virus  is  sufficient,  bub  in  times  of 
threatened  epidemic  two  or  more  inoculations  are  made  at  the  same  time, 
not  only  to  insure  a  successful  result,  but  rapidly  to  immunize  the 
patient.  It  would  appear  that  the  degree  of  immunity  bears  some  rela- 
tion to  the  number  or  size  of  the  vaccination  lesions,  and  this  can  readily 
be  understood  if  the  infection  is  local  and  the  body-cells  are  stimulated 
by  a  diffusible  toxin.  If,  however,  the  vaccination  lesion  is  but  the 
pointfof  entry  of  what  becomes  a  general  infection,  then  a  small  lesion 


FIG.  133. — VACCINIA  (SEVEN-DAY  LESION). 


FIG.  134. — VACCINIA  (NINE-DAY  LESION).          FIG.  135. — VACCINOID.     A  VAC- 
CINATION SCAR. 

Fig.  133  shows  a  seven-day  vaccination  vesicle.  Fig.  134  shows  a  nine-day 
vaccination  vesicle  just  before  pustulation  occurred.  Fig.  135  shows  a  recent  vacci- 
nation scar  with  pitting  and  radiation;  also  a  three-day  "vaccinoid"  or  "immunity 
reaction"  with  a  small  vesicle. 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  677 

should  suffice.  This  point  has  not  been  definitely  settled,  but  statistics 
tend  to  show  that  persons  vaccinated  in  two  or  more  areas  develop  an 
immunity  more  quickly  and  that  this  immunity  is  more  lasting. 

The  subsequent  care  of  the  wound  is  of  considerable  importance.  The 
operation  is  usually  regarded  as  a  trivial  one,  and  justly  so,  but  the 
lesion  requires  judicious  after-treatment  instead  of  being  entirely 
neglected,  as  it  so  often  is.  The  severe  infections  are  usually  attribut- 
able to  gross  and  careless  contamination  of  the  wound.  The  best  pos- 
sible protection  to  the  vaccinial  ulceration  is  afforded  by  the  forma- 
tion of  a  hard,  solid  crust,  due  to  desiccation  of  the  contents  of  the 
vaccine  vesicle  and  pustule.  Unless  undue  inflammation  and  suppura- 
tion set  in,  such  a  crust  will  form.  Care  must  be  taken  that  the  crust  is 
not  subjected  to  mechanical  violence  calculated  to  loosen  or  to  detach  it. 

Force  and  Stevens1  have  recently  advocated  the  routine  use  of  three 
inoculations  through  small  abrasions  made  with  a  chisel  2  mm.  in  diam- 
eter. This  method  is  regarded  as  resulting  in  greater  immunity  and 
less  likely  to  develop  secondary  infection. 

Constricting  shields  are  likely  to  be  unsatisfactory.  The  adhesion 
of  the  crust  to  the  sleeve  or  to  a  piece  of  protective  gauze  will  often 
lead  to  forcible  decrustation  when  the  sleeve  or  the  gauze  is  removed. 
Schamberg  and  Kolmer 2  have  found  that  daily  applications  of  a  4  per 
cent,  alcoholic  solution  of  picric  acid  upon  the  vaccinated  area  after 
the  first  forty-eight  hours  does  not  interfere  with  the  success  of  the 
vaccination,  and  lessens  the  degree  of  local  inflammatory  reaction  and 
constitutional  disturbances  by  hardening  the  epithelial  covering  of  the 
vaccine  lesion,  and  thereby  decreasing  the  liability  of  extraneous 
bacterial  infection. 

A  point  to  be  emphasized  is  that  severe  lesions  are  unnecessary,  and 
are  usually  due  to  scratching  of  the  vesicle  or  pustule  and  consequent 
introduction  of  dirt.  No  doubt  tetanus  bacilli  may  be  introduced  in 
this  manner,  the  resulting  scab  affording  the  necessary  anaerobic  con- 
ditions for  their  development. 

The  Phenomena  of  Vaccination;  Vaccinia. — Immediately  follow- 
ing vaccination  a  slight  redness  appears,  which  usually  subsides  rapidly. 
After  a  short  period  of  incubation — on  or  about  the  third  day — a  slight 
red  elevation  makes  its  appearance,  and  the  lesion  begins  to  burn  and 
itch.  On  the  sixth  or  seventh  day  the  abrasion  becomes  a  small,  silvery 
gray,  umbilicated  vesicle  with  a  sharply  raised  edge,  filled  with  a  clear 
serum,  and  surrounded  by  a  narrow  red  arepla  (Fig.  133).  By  the 

1  Jour.  Amer.  Med.  Assoc.,  1917,  Ixviii,  1247. 

2  The  Lancet,  London,  Nov.  18,  1911,  1397. 


678  ACTIVE    IMMUNIZATION 

tenth  day  the  characteristic  features  are  more  marked,  and  the  lesion  has 
usually  reached  its  height,  being  accompanied  by  a  burning  sensation 
and  an  almost  uncontrollable  desire  to  scratch  (Fig.  134).  The  areola 
is  now  quite  angry  in  appearance,  and  numerous  minute  vesicles  are 
seen  on  its  surface.  By  the  twelfth  day  the  areola  is  smaller,  the  con- 
tents become  turbid  and  commence  to  dry,  so  that  a  few  days  later  a 
scab  has  formed  that  drops  off  in  another  week  or  two. 

About  the  fifth  day  the  child  becomes  restless  and  irritable  and  shows 
a  slight  elevation  of  temperature.  These  symptoms  may  become  more 
pronounced  until  the  end  of  the  second  week,  when  they  subside  rapidly. 

Precautions  should  be  taken  to  prevent  scratching  and  infection  of 
the  vesicle.  The  old-time  "  beautiful  arms,"  with  well-marked  cellulitis 
and  adenitis  of  neighboring  glands,  were  largely  due  to  secondary  in- 
fection, and  are  not  at  all  necessary  in  the  process  of  vaccination. 
Evidence  would  tend  to  indicate  that  the  vesicle  is  the  typical  lesion 
of  both  smallpox  and  vaccinia,  and  that  the  pustules  are  simply  infected 
vesicles.  Ordinary  surgical  care  will  do  much  to  rob  vaccination  of  its 
discomfort  and  to  render  the  operation  a  most  harmless  one. 

The  results  of  a  vaccination,  therefore,  can  be  inspected  and  verified 
on  or  about  the  seventh  to  the  ninth  day.  With  persons  who  have  been 
vaccinated  successfully  on  a  previous  occasion  the  vaccinated  area  may 
show  a  slight  areola  at  the  end  of  twenty-four  hours,  with  or  without  a 
papule,  which  subsides  in  seventy-two  hours.  This  is  called  a  "reac- 
tion of  immunity,"  and  is  due  to  the  presence  of  antibodies  against  the 
virus.  Or  a  small,  itchy,  burning  papule  may  form,  which  develops 
into  a  small  vesicle  maturing  on  the  fifth  or  sixth  day,  and  then  rapidly 
subsiding,  constituting  the  reaction  known  as  vaccinoid  (Fig.  135). 
Occasionally  vaccination  is  followed  by  the  appearance  of  various 
eruptions. 

The  appearance  of  the  scar  varies  according  to  its  age  and  to  the 
degree  of  tissue  destruction.  The  physician  is  not  infrequently  re- 
quested to  examine  a  person  and  determine  if  the  scar  is  satisfactory 
evidence  of  successful  vaccination.  The  typical  good  scar  is  circular, 
and  about  the  size  of  a  ten-cent  piece,  with  smooth,  white,  and  depressed 
center  and  a  raised  border.  The  border  shows  numerous  radiations,  and 
the  entire  scar  may  show  little  pits  of  former  hair-follicles  when  the 
lesion  was  sufficiently  destructive  to  remove  the  upper  portion  of  the 
corium.  (See  Fig.  135.)  A  burn  or  an  ordinary  pyogenic  infection  may 
leave  scars  quite  similar  to  those  of  vaccination,  and  vaccination  scars 
may  show  wide  variation,  but  the  circumscribed  character,  the  raised 
border  with  radiations  and  depressions,  and  the  appearance  of  having 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  679 

been  stamped  on  the  skin  by  a  sharply  cut  die  are  quite  characteristic. 
Poor  scars  are  those  that  were  said  to  have  been  the  result  of  vaccination, 
but  in  very  many  instances  they  are  so  indistinct  as  to  make  it  difficult 
or  impossible  to  recognize  them  as  vaccination-marks. 

Revaccination. — One  successful  vaccination  does  not  necessarily 
confer  an  absolute  immunity  against  smallpox,  and  failure  to  recognize 
certain  limitations  in  this  respect  has  done  harm  by  enabling  anti- 
vaccinationists  to  create  a  distrust  in  the  minds  of  the  ignorant  by 
pointing  to  individual  instances  of  failure.  That  a  person  who  has 
once  been  vaccinated  may  afterward  suffer  from  smallpox  is  undoubted, 
but  usually  the  vaccination  was  performed  many  years  previously, 
and  in  any  case  the  disease,  when  it  does  occur,  is  relatively  mild  (vario- 
loid). 

There  can  be  no  doubt  but  that  the  immunity  gradually  diminishes. 
Perhaps  seven  years  may  be  taken  as  the  average  period  of  fairly  com- 
plete protection.  Children  should  be  vaccinated  within  the  first  year 
after  birth,  revaccinated  upon  entering  school,  and  again  after  leaving  it. 
If  smallpox  is  prevalent,  all  persons  should  be  vaccinated,  regardless 
of  the  fact  that  they  have  previously  been  vaccinated.  Only  those  who 
have  had  smallpox  may  be  excused.  If,  as  a  matter  of  fact,  persons  are 
still  immune,  the  vaccination  will  not  "take"  and  no  harm  is  done, 
whereas  if  it  succeeds,  such  persons  will  have  the  satisfaction  of  knowing 
that  their  immunity  has  been  increased.  Hence  it  cannot  be  too 
strongly  emphasized  that  not  only  vaccination,  but  revaccination,  is 
indicated  to  protect  the  individual  and  society  against  smallpox.  Dwyer 
claims  that  a  person  should  be  revaccinated  repeatedly  in  succession 
until  he  fails  to  react;  even  a  slight  "take"  would  indicate  incomplete 
immunity. 

Occasionally  a  non-immunized  person  refuses  to  "take,"  but  vac- 
cination should  be  repeated  three  or  four  times,  as  failure  is  not  infre- 
quently due  to  old  and  inactive  virus,  and  the  actual  number  of  persons 
absolutely  insusceptible  is  very  small  indeed. 

Risks  of  Vaccination. — When  vaccination  is  properly  performed  with 
a  good  virus,  the  risk  of  permanent  injury  to  life  or  limb  is  almost 
negligible. 

1 .  Tetanus. — The  most  serious  of  the  inj  uries  that  have  been  attributed 
to  vaccination  is  tetanus.  The  tetanus  bacillus  and  its  spores  are  so 
wide-spread  in  nature  that  opportunity  presents  itself  for  contamination 
of  the  vaccinal  wound  and  the  virus  itself.  Every  precaution  should, 
therefore,  be  taken  in  the  preparation  of  virus,  and  it  is  especially  im- 
portant that  physicians  and  laymen  should  realize  the  necessity  for 


680  ACTIVE    IMMUNIZATION 

observing  ordinary  care,  and  at  least  ordinary  cleanliness,  in  the  treat- 
ment of  the  vaccinal  wound. 

It  is  exceedingly  difficult  to  determine  the  source  of  infection  in  each 
case  of  vaccinal  tetanus,  but  experimental  investigations  would  tend 
to  indicate  that  the  virus  itself  is  seldom,  if  ever,  the  vehicle  of  infection. 
John  F.  Anderson,  Director  of  the  Hygienic  Laboratory,  in  testimony 
given  before  the  Pennsylvania  State  Vaccination  Commission,  stated 
that  in  experiments  carried  out  on  monkeys  and  guinea-pigs  with  vaccine 
lymph  purposely  contaminated  in  the  laboratory  with  countless  numbers 
of  tetanus  spores,  it  was  found  impossible  to  communicate  tetanus 
in  this  manner,  although  the  vaccinations  were  more  severe  than  the 
ordinary  vaccinations  performed  on  man,  in  that  several  places  were 
inoculated  and  the  areas  abraded  were  large.  Anderson  stated  that 
his  "  conclusion  from  these  experiments  is  that  it  is  almost  impossible 
to  produce  tetanus,  even  with  vaccine  virus  that  contains  tetanus 
germs  in  it,  by  the  simple  act  of  vaccination." 

Since  1909  there  were  approximately  100,000  specimens  of  vaccine 
virus  examined  in  the  Hygienic  Laboratory,  particularly  with  the  pur- 
pose of  determining  the  presence  of  tetanus  germs  or  their  products. 
The  vaccine  was  purchased  in  the  open  market,  and  the  examinations 
were  made  as  thoroughly  as  it  was  possible  to  make  them.  To  use 
Anderson's  words:  "  We  have  never  succeeded  in  finding  any  evidence 
of  the  presence  of  the  tetanus  organism  or  its  products  in  vaccine  virus." 
(Report  of  the  Commission.) 

It  would  appear,  therefore,  that  virus  prepared  according  to  modern 
methods  and  with  all  recognized  precautions  is  safe.  In  view  of  the 
incidence  of  tetanus  following  other  injuries,  it  is  reasonable  to  conclude 
that  most  cases  of  vaccinal  tetanus  are  secondary  wound  infections, 
and  therefore  largely  preventable. 

2.  Syphilis. — With  the  use,  years  ago,  of  humanized  virus,  and  par- 
ticularly in  the  days  of  arm-to-arm  vaccination,  extremely  rare  instances 
of  the  transmission  of  syphilis  have  been  known  to  occur.     Since,  how- 
ever, the  use  of  calf  virus,  which  is  the  virus  exclusively  employed  in  this 
country,  such  an  accident  is  absolutely  impossible,  as  calves  are  not 
susceptible  to  luetic  disease. 

3.  Cancer,  foot  and  mouth  disease,  tuberculosis,  and  various  chronic 
skin  eruptions  have  been  attributed  to  vaccination  by  its  opponents; 
none  of  these  claims  has,  however,  been  substantiated. 

Protective  Value  of  Vaccination. — Of  the  value  of  the  protection 
afforded  by  vaccination  against  smallpox  there  can  be  no  doubt  in  the 


PROPHYLACTIC    IMMUNIZATION   OR   VACCINATION  681 

minds  of .  right-thinking  and  unbiased  persons.  The  history  of  the 
world  before  the  days  of  universal  vaccination  shows  the  wide  prevalence 
of  smallpox  and  its  fearful  mortality.  It  was  regarded  as  a  disease  of 
childhood,  owing  to  the  fact  that  all  contracted  it  at  the  earliest  op- 
portunity, and,  accordingly,  smallpox  was  the  cause  of  a  fearful  infant 
mortality. 

At  the  present  day,  owing  to  the  general  employment  of  vaccination, 
smallpox  is  a  rare  disease,  but  its  very  rarity  has  fostered  a  certain  de- 
gree of  false  security  and  carelessness  in  carrying  out  the  process.  A 
young  and  new  generation  of  non-vaccinated  persons  in  any  community 
is  a  source  of  danger,  and  accordingly  sporadic  cases  are  often  blessings 
hi  disguise,  from  the  fact  that,  when  they  appear,  compulsory  vaccina- 
tion is  then  instituted  and  large  numbers  seek  revaccination. 

In  Germany,  where  vaccination  is  compulsory,  smallpox  is  now  a 
comparatively  rare  disease.  While  the  general  death-rate  from  all 
diseases  is  lower  in  England  and  Wales  than  in  Germany,  the  smallpox 
mortality  is  seven  and  one-half  times  the  mortality  of  Germany,  and, 
proportionate  to  the  population,  over  13  times. 

Austria,  one  of  Germany's  neighbors,  had,  for  the  twenty  years 
following  1874,  almost  30  times  as  high  a  smallpox  mortality  as  Germany. 
During  this  period  239,800  persons  perished  in  Austria  from  smallpox 
alone. 

Physicians  should  carefully  impress  upon  those  over  whom  they  have 
any  influence  the  necessity  of  being  vaccinated,  for  only  a  thoroughly 
vaccinated  population  can  solve  the  problem  of  exterminating  smallpox 
as  an  epidemic  disease. 

RABIES 

There  are  but  few  diseases  more  dreaded  by  the  laity  than  rabies, 
or  hydrophobia.  Tales  of  the  sufferings  of  infected  persons,  especially 
those  with  the  furious  variety  of  this  infection,  characterized  by  maniacal 
symptoms  and  dread  of  water  (hydrophobia),  have  been  thoroughly 
disseminated,  so  that  the  cry  of  "mad  dog!"  on  the  public  streets  is 
sufficient  to  arouse  a  general  state  of  hysteric  excitement  in  which  an 
otherwise  harmless  creature  may  be  compelled  to  bite  or  snap  for  self- 
protection.  Not  all  dogs  under  these  conditions  are  mad  or  infected 
with  rabies,  and  the  bite  of  an  angry  dog,  otherwise  normal,  is  not 
necessarily  dangerous  from  the  standpoint  of  rabic  infection.  However, 
almost  every  one,  upon  being  bitten  by  a  dog,  will  promptly  consult 
his  physician,  and  this  is  proper  and  to  be  encouraged.  Genuine  rabies 


682  ACTIVE    IMMUNIZATION 

is  an  acute  infectious  disease  in  which  the  diagnosis  is  quite  readily 
made,  and  whenever  possible  an  effort  should  be  made  either  to  confirm 
or  to  disprove  the  diagnosis  by  making  an  examination  of  the  animal's 
brain,  and  if  the  dog  is  found  to  have  been  free  from  rabies,  this  fact 
should  be  carefully  impressed  upon  the  patient,  as  otherwise  the  dread 
of  infection  may  weigh  heavily  upon  the  patient  and  lead  to  distressing 
nervous  disturbances. 

While  the  infectiousness  of  rabies  has  been  known  for  a  great  many 
years  and  was  proved  experimentally  by  Galateir1  and  Pasteur,2  it  was 
not  until  Negri,  in  1903,  described  certain  bodies  (Negri  bodies),  seen 
by  him  in  large  nerve-cells  in  sections  of  the  central  nervous  system, 
that  anything  was  found  that  seemed  absolutely  specific  for  rabies. 
Negri  regarded  these  bodies  as  specific  for  rabies  and  probably  of  a 
protozoan  nature.  Later  investigations  fully  established  the  diagnos- 
tic value  of  these  bodies,  and  their  definite  characteristic  morphology, 
evidences  of  cyclic  development,  and  staining  qualities  indicate  a 
protozoan  structure  resembling  members  of  the  Rhizopoda,  designated 
by  Anna  Williams  in  19063  as  Neurorrhyctes  hydrophobia. 

Rembringer,4  Poor  and  Steinhardt,5  Bertarelli  and  Volpino6  have 
demonstrated  the  filterability  of  the  rabic  virus,  and  Noguchi7  has  cul- 
tivated from  both  " street"  and  "fixed"  virus,  very  minute  granular 
and  somewhat  coarser  pleomorphic  chromatoid  bodies  which,  on  sub- 
sequent transplantation,  reappeared  in  the  new  cultures  through  many 
generations  and  reproduced  typical  symptoms  of  rabies  in  dogs,  rabbits, 
and  guinea-pigs. 

To  Pasteur  is  due  the  credit  for  having  discovered  (1880)  the  fact 
that  the  disease  may  be  prevented  by  conferring  gradual  immunization 
with  increasing  doses  of  the  attenuated  virus.  This  treatment,  with 
some  modification,  is  now  used  with  evident  success  in  all  parts  of  the 
world. 

Nature  of  Rabies. — The  virus  or  parasite  is  contained  in  the  saliva 
of  the  rabid  animal,  and  infection  is  possible  when  the  skin  is  abraded 
by  bites  and  scratches.  The  virus  travels  by  way  of  the  nerve-paths 
to  the  central  nervous  tissue,  and,  as  in  tetanus,  the  symptoms  of  the 
disease  are  due  to  involvement  of  these  tissues. 

1  Compt.  rend.  Acad.  d.  sc.,  1879,  Ixxxix,  444. 

2  Compt.  rend.  Acad.  d.  sc.,  1881,  xcii,  159. 

3  Proc.  N.  Y.  Path.  Soc.,  1906,  vi,  77. 

4  Ann.  de  PInst.  Pasteur,  1903,  xvii,  834;  1904,  xviii,  150. 
6  Jour.  Infect.  Dis.,  1913,  xii,  202. 

6  Centralbl.  f.  Bakt.,  Orig.,  1904,  xxxvii,  51.     Bertarelli,  ibid. 

7  Jour.  Exper.  Med.,  1913,  xviii,  314. 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  683 

The  period  of  incubation,  or  the  time  elapsing  between  the  time  of 
injury  and  the  first  symptoms,  is  quite  variable,  ranging  from  twenty 
to  sixty  days,  although  it  may  be  as  short  as  ten  days.  As  in  tetanus, 
this  period  depends  upon — (a)  the  location  of  the  injury;  (6)  the  quantity 
or  dose  of  virus ;  (c)  the  kind  of  animal  responsible  for  the  injury.  Bites 
about  the  face  and  fingers,  especially  if  they  are  deep  and  lacerating, 
are  especially  dangerous;  bites  about  the  back  and  lower  limbs,  espe- 
cially if  superficial,  are  much  less  dangerous,  and  accompanied  by  a 
longer  period  of  incubation.  It  is  to  be  remembered  that  bites  may  be 
infectious  as  early  as  nine  days  before  the  dog  shows  well-marked  symp- 
toms of  the  disease.  Not  infrequently  an  animal  is  observed  to  be 
surly  and  snappy  for  several  days  before  rabid  symptoms  develop,  and 
a  bite  during  this  time  should  be  regarded  as  dangerous. 

Only  about  16  per  cent,  of  human  beings  bitten  by  rabid  animals  and 
untreated  appear  to  contract  rabies.  Since  the  establishment  of  the 
Pasteur  treatment  of  the  disease,  the  percentage  of  developed  cases 
after  bites  is  much  lower — about  0.46  per  cent. 

Diagnosis  and  Management  of  Rabies. — Even  though  an  animal 
is  unmistakably  rabic,  every  effort  should  be  made  to  destroy  the  animal, 
not  only  in  order  to  prevent  further  damage,  but  to  corroborate  the 
diagnosis  by  microscopic  examination  of  the  nervous  tissues  for  Negri 
bodies. 

1.  As  a  general  rule,  all  animal  bites. should  receive  surgical  attention. 
Wounds  produced  by  animals  clinically  rabid  should  be  cauterized  at 
once  with  fuming  nitric  acid  or  pure  phenol.     This  is  done  to  offset 
the  delay  in  securing  the  Pasteur  treatment,  and  because  there  is  evi- 
dence to  show  that  thorough  cauterization  of  the  wound  is  in  itself 
highly  beneficial. 

2.  The  animal  should  be  promptly  destroyed,  not  only  to  prevent 
further  damage,  but  in  order  to  make  a  microscopic  diagnosis  by  ex- 
amination of  the  brain  for  Negri  bodies.     This  examination  is  highly 
important  and  should  never  be  omitted,  for  if  it  shows  the  absence  of 
bodies,  this  fact  should  be  carefully  impressed  upon  the  patient,  as 
there  is  no  doubt  that  a  neurotic  element,  amounting  in  many  instances 
to  actual  hysteria,  may  cause  considerable  harm  to  the  patient  even 
though  he  is  definitely  free  from  rabic  infection. 

3.  The  whole  dog  may  be  packed  in  ice  and  shipped  at  once  to  a 
central  laboratory,  or  the  head  alone  may  be  removed  and  packed  in 
ice  or  glycerin  and  promptly  shipped.     The  brain  should  not  be  dis- 
turbed.   When  it  reaches  the  laboratory,  the  diagnosis  should  be  made 
at  once  by  " smear"  preparations  and  sections  of  the  brain  demonstrat- 


684  ACTIVE    IMMUNIZATION 

ing  the  characteristic  Negri  bodies  in  the  large  ganglion-cells,  and  con- 
firmed by  inoculating  emulsions  of  the  brain  into  guinea-pigs  or  rabbits. 
The  latter  requires  from  ten  to  twenty  days  before  the  result  may  be 
known.  A  negative  animal  inoculation  test  is  better  evidence  than  a 
negative  smear  or  section;  obviously,  these  examinations  are  to  be 
made  only  by  properly  trained  persons. 

According  to  Park,  the  value  of  the  smear  method  of  diagnosis  may 
be  summarized  as  follows: 

1.  Negri  bodies  demonstrated,  diagnosis,  rabies. 

2.  Negri  bodies  not  demonstrated  in  fresh  brains,  very  probably 

not  rabies. 

3.  Negri  bodies  not  demonstrated  in  decomposing  brains,  uncer- 

tain. 

4.  Suspicious  bodies  in  fresh  brains,  probably  rabies. 

4.  If  the  animal  was  clinically  rabid,  the  Pasteur  treatment  should 
be  commenced  as  soon  as  possible,  without  waiting  for  the  laboratory 
report,  if  this  will  be  delayed  for  several  days.     Wounds  about  the  face 
and  hands,  where  there  is  no  clothing  to  retain  the  infectious  saliva, 
should  receive  intensive  treatment;    otherwise  the  milder  course  of 
immunization  will  suffice. 

5.  As  a  general  rule,  it  is  well  to  send  the  patient  to  a  regular  Pasteur 
institute,  where  there  are  special  facilities  for  the  proper  treatment  of 
these  cases.     Otherwise  the  physician  may  treat  the  patient  at  home. 
Several  large  manufacturing  firms  are  prepared  to  ship  by  mail  the  fresh 
daily  treatments  properly  preserved  and  ready  for  administration. 

6.  The  Pasteur  treatment  should  be  given  to  every  patient  bitten 
by  a  rabid  animal  or  by  one  suspected  of  being  rabid.     Not  all  persons 
are  necessarily  infected,  even  by  bites  of  rabid  animals,  but  this  should 
not  unduly  influence  the  physician,  for  he  will  not  have  fulfilled  his  duty 
unless  he  carefully  explains  the  etiology  of  the  disease  and  advises  im- 
mediate immunization.     Aside  from  the  actual  benefits  of  the  treat- 
ment, the  mental  effect  upon  the  patient  is  deserving  of  consideration. 
Even  slight  wounds  by  rabid  animals,  wherever  their  location,  should 
be  regarded  as  dangerous,  and  the  Pasteur  treatment  advised  in  addi- 
tion to  routine  cauterization. 

7.  With  severe  bites  of  angry  but  not  necessarily  clinically  rabid 
dogs,  the  treatment  depends  upon  various  factors.     In  any  case  the 
wound   should   be   thoroughly   cauterized   and   the   animal   carefully 
guarded  (not  killed)  for  two  or  three  weeks.     If  rabid  symptoms  appear, 
the  animal  should  be  destroyed,  the  cerebral  tissues  examined,  and  the 
Pasteur  treatment  of  the  patient  begun. 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  685 

8.  All  animals  bitten  by  a  rabid  animal  should,  of  course,  be  promptly 
destroyed;  even  those  bitten  by  a  dog  suspected  of  being  rabid  should 
be  destroyed  or  closely  guarded  until  a  definite  diagnosis  can  be  reached. 
In  England,  where  strict  laws  are  enforced  relative  to  the  muzzling  and 
control  of  dogs,  rabies  is  relatively  infrequent,  and  it  is  especially  urged 
that  similar  measures  be  adopted  and  enforced  in  our  own  communities, 
particularly  during  the  summer  months. 

Principle  of  the  Pasteur  Treatment  (Active  Immunization)  of  Rabies. 
— This  method  is  based  upon  the  principle  of  stimulating  the  production 
of  rabic  antibodies  by  injecting  attenuated  or  modified  virus  during  the 
period  of  incubation,  so  that  the  virus  introduced  into  the  wound  is 
destroyed,  neutralized,  or  its  effects  neutralized,  while  the  virus  itself 
is  finally  destroyed.  Pasteur  worked  out  this  theory  and  established 
its  truth  by  experiments  upon  the  lower  animals  before  applying  the 
treatment  to  man. 

By  passing  the  virus  through  a  series  of  rabbits,  the  period  of  incuba- 
tion is  shortened  to  about  six  to  seven  days,  and  at  the  same  time  its 
pathogenicity  for  man  is  actually  diminished  (virus  fixe).  By  drying 
the  tissues  containing  the  fixed  virus  attenuation  is  secured,  so  that  it 
is  easily  possible  so  to  modify  the  virus  that  it  cannot  produce  rabies  in 
man,  but  yet  is  able  to  produce  the  specific  antibodies.  The  Pasteur 
treatment  is,  therefore,  a  process  of  active  immunization  with  emul- 
sions of  a  tissue  (spinal  cords  of  infected  rabbits)  in  which  the  virus 
has  been  attenuated  by  a  process  of  drying  and  desiccation.  The  early 
doses  consist  of  highly  attenuated  cords,  and  succeeding  doses  become 
gradually  more  potent,  as  is  usual  in  the  technic  of  any  method  of  active 
immunization. 

Preparation  of  the  Rabies  Vaccine. — As  a  preliminary,  it  is  necessary 
to  prepare  or  obtain  "virus  fixe.''  This  may  generally  be  procured 
from  a  laboratory,  or  may  be  prepared  by  passing  street  virus  from  the 
medulla  of  a  rabic  cow  or  dog  through  a  series  of  young  rabbits.  After 
from  30  to  50  passages  the  incubation  period  is  gradually  reduced  to  six 
or  eight  days  ("  virus  fixe") . 

1.  From  an  animal  succumbing  the  day  or  night  before,  a  piece  of 
the  floor  of  the  fourth  ventricle  measuring  about  2  cm.  in  length  is 
emulsified  in  1  c.c.  of  sterile  bouillon,  and  three  or  four  drops  of  this 
emulsion  are  injected  beneath  the  dura  of  a  normal  rabbit.  In  large 
institutes  two  or  more  rabbits  are  injected  daily.  The  inoculation  is 
quickly  and  easily  performed  by  trephining  a  small  area  in  the  median 
line  of  the  forehead  and  injecting  the  emulsion  beneath  the  dura  mater 


686 


ACTIVE    IMMUNIZATION 


with  a  syringe.     The  whole  operation  must  be  carried  out  in  an  aseptic 
and  practically  painless  manner. 

2.  After  inoculation  the  animals  are  placed  in  clean  cages;  in  from 
six  to  eight  days  paralytic  symptoms  of  rabies  appear,  followed  in  three 
to  four  days  by  death.  The  hair  is  then  sprayed 
with  a  solution  of  lysol  and  the  skin  removed. 
The  cord  and  brain  are  then  extracted  under 
aseptic  precautions.  The  cord  is  severed  just 
below  the  medulla,  a  portion  is  snipped  off  into 
sterile  bouillon  for  culture,  and  then  divided  into 
two  equal  pieces  which  are  suspended  by  sterilized 
silk  threads  in  a  sterile  glass  jar  containing  flakes 
of  caustic  potash  (Fig.  136).  The  medulla  is 
placed  in  a  sterile  dish,  and  is  used  to  continue 
the  inoculations,  as  was  previously  described.  A 
postmortem  examination  is  finally  performed,  and 
any  cord  in  which  the  animal  is  found  diseased  or 
in  which  the  culture  of  the  cord  shows  bacterial 
contamination  is  rejected. 

The  jars  with  suspended  cords  are  kept  in  a 
special  room  at  a  temperature  of  about  20°  to  25°  C. 
3.  After  a  suitable  period  of  drying  pieces  of 
cord  are  prepared  for  injection.  This  is  performed 
in  various  ways  at  different  laboratories;  no  at- 
tempt at  exact  dosage  is  made.  In  the  New  York 
Board  of  Health  laboratories  1  cm.  of  the  cord  is 
thoroughly  emulsified  in  3  c.c.  of  sterile  saline 
solution,  the  process  being  conducted  in  an  aseptic 
manner.  If  the  material  is  to  be  shipped,  an 
addition  of  20  per  cent,  of  glycerin  and  0.5  per  cent,  of  phenol  is 
made. 

Administration  of  the  Vaccine. — Injections  are  given  with  a  sterile 
syringe.  The  abdominal  region  of  the  patient  is  bared,  a  spot  touched 
with  tincture  of  iodin,  wiped  with  alcohol,  and  the  injection  given  sub- 
cutaneously.  Keirle  does  not  vary  the  dose  according  to  the  age,  both 
the  old  and  the  young  receiving  the  same  dose.  At  times  the  injection 
is  followed  by  redness  and  induration  in  the  subcutaneous  tissues,  but 
abscesses  are  never  formed. 

Cases  of  severe  injury,  such  as  deep  bites  about  the  face  and  fingers, 
should  be  rapidly  immunized  (intensive  treatment);  in  other  cases  the 


FIG.  136.— PREPARA- 
TION OF  RABIES 
VACCINE. 
Note   the    cords 
suspended  within  the 
jar  by  means  of  sterile 
silk  threads;  sticks  of 
sodium   hydroxid    to 
absorb  moisture  and 
hasten  desiccation. 


PROPHYLACTIC    IMMUNIZATION    OR   VACCINATION 


687 


treatment  may  be  mild  (mild  treatment).  The  uniform  dose  of  cord 
emulsion,  prepared  as  just  described,  is  2.5  c.c.  The  series  of  inocula- 
tions given  in  the  Research  Laboratory  of  New  York  in  treating  human 
cases  after  an  average  bite  are  as  follows: 

TABLE   23— SCHEDULE   OF   INOCULATIONS   IN   IMMUNIZATION 

AGAINST  RABIES 


DAY 

MILD  TREATMENT 

INTENSIVE  TREATMENT 

First 

14  and  13  day  cord 

12  and  11  day  cord.     Repeated  in  p.  M. 

Second 

12  and  11  day  cord 

10  and    9  day  cord;  8  and  7  day  cord  p.  M. 

Third 

10  and    9  day  cord 

6  day  cord 

Fourth 

8  and    7  day  cord 

5  day  cord 

Fifth      

6  day  cord 

4  day  cord 

Sixth   

5  day  cord 

3  day  cord 

Seventh  

4  day  cord 

2  day  cord 

Eighth  

3  day  cord 

4  day  cord 

Ninth 

2  day  cord 

4  day  cord 

Tenth 

4  day  cord 

1  day  cord 

Eleventh 

3  day  cord 

4  day  cord 

Twelfth  
Thirteenth    . 

2  day  cord 
4  day  cord 

3  day  cord 
2  day  cord 

Fourteenth  
Fifteenth  

5  day  cord 
2  day  cord 

4  day  cord 
1  day  cord 

Sixteenth  
Seventeenth  
Eighteenth 

4  day  cord 
3  day  cord 
2  day  cord 

4  day  cord 
3  day  cord 
2  day  cord 

Nineteenth  
Twentieth      .... 

4  day  cord 
3  day  cord 

4  day  cord 
3  day  cord 

Twenty-first  

2  day  cord 

2  day  cord 

Results. — According  to  reliable  statistics,  the  mortality  of  rabies 
without  the  Pasteur  treatment  is  about  16  per  cent.;  with  the  treat- 
ment the  average  mortality  is  about  0.46  per  cent.  The  mortality  of 
those  bitten  about  the  face  or  head  is  about  1.25  per  cent.;  of  those 
bitten  on  the  hand,  0.75  per  cent. ;  of  those  bitten  on  other  parts  of  the 
body,  a  little  over  0.25  to  1  per  cent.  In  the  Pasteur  Institute  of  Paris 
only  such  persons  are  treated  as  have  been  lacerated,  so  that  the  virus 
has  gained  entry  into  the  wounds.  Viala1  reports  that  during  the  year 
1915  as  many  as  654  persons  were  treated,  with  a  single  death.  Taking 
into  consideration  only  those  cases  in  which  the  diagnosis  of  rabies 
has  been  confirmed  in  the  animal  by  a  competent  examiner,  the  mortality 
of  the  cases  treated  at  the  Pasteur  Institute  in  Paris  for  the  past  ten 
years,  and  covering  the  treatment  of  nearly  6000  persons,  is  only  0.6  per 
cent.,  which,  compared  to  the  average  mortality  of  16  per  cent,  without 
vaccine  treatment,  speaks  most  favorably  for  the  value  of  Pasteur's 
antirabic  immunization. 

The  bites  of  wolves  are  more  fatal  than  those  of  dogs,  the  mortality 
1  Ann.  de  1'Last.  Pasteur,  1916,  xxx,  422. 


688  ACTIVE    IMMUNIZATION 

being  about  10  per  cent,  in  spite  of  the  intensive  treatment,  and  about 
40  to  60  per  cent,  without  treatment. 

When  symptoms  of  rabies  have  appeared,  the  treatment  is  unavail- 
ing. Antirabic  serums  have  been  prepared  by  immunizing  animals, 
such  as  sheep  and  horses,  and  these  should  be  tried  in  human  patients 
presenting  symptoms,  but  the  results  in  general  have  not  been  uniformly 
encouraging. 

TYPHOID  FEVER 

Our  knowledge  of  vaccination  in  typhoid  fever  begins  with  the  work 
of  Pfeiffer  and  Kolle.1  These  observers,  in  1896,  immunized  two  volun- 
teers with  heat-killed  cultures,  and  by  complete  laboratory  investiga- 
tions demonstrated  the  identity  of  the  immunity  following  an  attack  of 
the  disease  with  the  artificial  immunity  produced  by  inoculation.  At 
about  the  same  time  Wright,2  of  London,  inoculated  two  men  with 
killed  cultures,  and  a  year  later  published  the  results  of  the  successful 
vaccination  of  17  persons.  In  1898  be  continued  the  work  in  India, 
where  4000  soldiers  were  inoculated,  with  encouraging  results.  Later, 
during  the  Boer  war,  Wright  and  Leishman  treated  100,000  men,  and 
the  results,  while  good,  were  not  encouraging,  due,  as  pointed  out  later 
by  Leishman,3  to  the  fact  that  the  vaccine  was  damaged  during  its 
preparation  by  overheating.  Since  1904  an  improved  vaccine  has  been 
used  among  the  British  troops  in  India  in  ever-increasing  quantities, 
with  uniformly  good  results. 

Antityphoid  vaccination  was  begun  in  the  United  States  army  in 
1908,  the  vaccine  being  prepared  by  Major  Frederick  F.  Russel.  Its 
value  has  been  established  so  clearly  that  vaccination  is  now  compulsory. 
The  results  obtained  in  the  army  have  had  considerable  influence  in 
establishing  a  wide-spread  general  confidence  in  antityphoid  inoculation. 

Preparation  of  Typhoid  Vaccine. — Based  upon  general  principles, 
the  vaccine  should  be  prepared  of  typhoid  bacilli  as  little  changed  by 
heat  or  chemicals  as  possible.  Russel  has  prepared  the  army  vaccine 
with  a  single  avirulent  culture  which  proved  by  animal  experiments 
and  laboratory  methods  capable  of  producing  large  quantities  of  immune 
agglutinins  and  bacteriolysins.  As  a  general  rule,  however,  the  vaccine 
should  be  polyvalent,  and  particularly  in  view  of  the  experiments  of 
Hooker,4  who  has  shown  by  complement-fixation  tests  consistent  anti- 
genie  differences  among  some  strains  of  Bacillus  typhosus. 

1  Deutsch.  med.  Wchnschr.,  1896,  xxii,  735. 

2  The  Lancet,  London,  September  19,  1896,  907;  Brit.  Med.  Jour.,  January  30, 
1897,  16. 

3  Jour.  Roy.  Inst.  4  Jour.  Immunology,  1916,  11,  1. 


PROPHYLACTIC    IMMUNIZATION   OR   VACCINATION  689 

The  preparation  of  the  vaccine  is  comparatively  simple.  The  bacilli 
are  grown  on  agar  for  twenty-four  hours,  washed  off  with  sterile  normal 
salt  solution,  standardized  by  counting  the  bacilli,  and  killed  by  heating 
to  56°  C.  for  one  hour.  As  a  matter  of  safety,  0.25  per  cent,  of  tricresol 
is  then  added  (Russel).  The  details  of  the  technic  are  given  in  Chapter 
XIII. 

Metchnikoff  has  never  fully  accepted  the  belief  in  the  value  of  heat- 
killed  vaccines,  and  at  present  is  actively  concerned  with  vaccines  pre- 
pared of  living  bacilli  sensitized  with  their  immune  serum  (sensitized 
vaccines).  Injection  of  these  vaccines  into  chimpanzees  is  not  followed 
by  any  untoward  effects,  and  apparently  the  bacilli  so  administered  are 
destroyed  at  once,  as  they  have  not  been  found  in  the  blood,  urine,  and 
feces.  Metchnikoff  and  Besredka  have  immunized  persons  according 
to  these  methods  and  report  excellent  results.  Obviously,  there  is  some 
reluctance  in  using  a  vaccine  of  living  bacilli  until  extended  animal 
experiments  have  proved  that  they  are  harmless  and  more  efficient  than 
the  vaccines  of  killed  bacilli. 

As  a  general  rule,  the  vaccine  is  prepared  in  two  strengths:  500,000,- 
000  bacteria  per  cubic  centimeter  for  the  first  dose,  and  1,000,000,000  for 
the  second  and  third  doses. 

Method  of  Inoculation. — The  vaccine  is  best  administered  at  about 
4  o'clock  in  the  afternoon,  so  that  the  reaction  appears  during  the  night 
and  is  least  likely  to  be  disturbing.  It  is  well  to  administer  a  cathartic 
the  day  before  the  inoculation  is  made.  Inoculations  should  not  be 
given  during  the  menstrual  period,  as  the  general  reaction  is  likely  to  be 
somewhat  severer  at  this  time. 

The  skin  over  the  insertion  of  the  deltoid  muscle  is  touched  with 
tincture  of  iodin  and  the  injection  given  subcutaneously.  Intramuscular 
injections  should  be  avoided,  as  the  reactions  are  more  unpleasant  and 
accompanied  by  unnecessary  pain  on  movement.  In  making  deep 
injections  there  is  also  danger  of  striking  a  nerve,  a  proceeding  that  may 
be  followed  by  disagreeable  neuritis. 

After  the  injection  has  been  given  the  iodin  is  wiped  away  with  a 
pledget  of  cotton  and  alcohol;  no  dressing  is  necessary. 

The  syringe  and  needle  should  be  sterile.  Commercial  firms  have 
placed  the  prophylactic  on  the  market  in  syringes  with  sterilized  needles, 
accompanied  with  full  instructions  as  to  the  technic  of  administration. 
The  vaccine  should  be  well  shaken  before  the  injection  is  given. 

When  a  large  number  of  injections  are  to  be  given  at  one  time,  a 
single  syringe  may  be  used  with  a  large  number  of  sterile  needles,  a 
44 


690 


ACTIVE    IMMUNIZATION 


separate  needle  being  used  for  each  person.     The  vaccine  may  be  put 
up  in  individual  ampules  or  in  a  stock  bottle,  the  former  being  preferable. 

Dosage. — Three  injections  are  given  at  intervals  of  one  week.     For 

adults  (150  pounds)  the  doses  used  in  the  army  have  been  as  follows: 

First  dose:          500,000,000  bacilli. 

Second  dose:    1,000,000,000  bacilli. 

Third  dose:      1,000,000,000  bacilli. 

These  amounts  are  contained  in  1  c.c.  (about  15  minims).  Children 
receive  doses  in  proportion  to  their  weight;  if  the  dose  cannot  be  divided 
evenly,  it  is  better  to  give  a  little  more  rather  than  a  little  less,  for  chil- 
dren tolerate  the  injections  remarkably  well. 

Reactions. — Persons  in  poor  physical  condition  are  more  likely  than 
the  robust  to  experience  disagreeable  after-effects. 

The  local  reaction  consists  of  a  small  red  and  tender  area  lasting  about 
forty-eight  hours.  Occasionally  the  edema  and  pain  may  be  more 
marked,  but  abscess  formation  is  practically  unknown. 

The  general  reaction,  when  present,  gives  rise  to  headache,  malaise, 
and  sometimes  to  fever,  chills,  and  occasionally  to  nausea,  vomiting,  or 
diarrhea.  The  severe  reactions  are  not  alarming  and  disappear  quickly. 

The  inoculated  person  should  abstain  from  severe  exercise  for  the 
following  twenty-four  hours  and  rest;  in  the  great  majority  of  instances 
our  soldiers  have  not  been  inconvenienced  and  were  able  to  continue 
with  their  routine  duties. 

The  following  tables,  compiled  by  Major  Russel,1  show  the  propor- 
tion of  reactions  in  adults  and  children: 

TABLE  24.— PERCENTAGE  OF  GENERAL  REACTIONS  IN  ADULTS 

(128,903  DOSES) 


DOSE 

NONE 

MILD 

MODERATE 

SEVERE 

First  

68.2 

28.9 

2.4 

0.3 

Second  

71.3 

25.7 

2.6 

0.2 

Third  

78.0 

20.3 

1.5 

0.1 

TABLE  25.—PERCENTAGE  OF  GENERAL  REACTIONS  IN  359  CHILDREN, 
TWO  TO  SIXTEEN  YEARS  OF  AGE 


DOSE 

NONE 

MILD 

MODERATE 

SEVERE 

First 

73.54 

24.51 

1.67 

0.28 

Second  
Third  

86.26 
92.56 

11.99 
0.38 

1.75 
1.06 

1  Jour.  Amer.  Med.  Assoc.,  1913,  Ix,  344. 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION  691 

A  comparison  of  these  tables  shows  that  the  general  reaction  is  much 
more  infrequent  or  milder  in  children  than  in  adults,  even  after  the  first 
dose;  after  the  second  and  third  doses  the  difference  is  more  marked. 

In  former  years  considerable  stress  was  laid  upon  the  possibility  of 
a  negative  phase  following  the  inoculation,  during  which  a  person  was 
believed  to  be  more  susceptible  to  infection.  This  is  now  believed  by 
Leishman,  Russel,  and  others  of  extended  experience  to  be  incorrect, 
the  more  general  belief  being  that  inoculations  may  be  made  and  are 
especially  indicated  during  epidemics  of  the  disease. 

Duration  and  Degree  of  Typhoid  Immunity. — It  should  be  empha- 
sized that  immunity  following  typhoid  immunization  is  not  absolute,, 
and  an  immunized  person  cannot  afford  to  neglect  ordinary  precautions- 
against  infection.  A  lowered  state  of  general  body  health  or  a  large  dose 
of  infectious  material  may  at  any  time  result  in  infection. 

The  prophylactic  treatment  should  be  used  in  conjunction  with 
well-known  sanitary  precautions  in  order  to  obtain  the  best  results. 

The  immunity  is  apparently  manifest  soon  after  the  first  and  second 
doses  have  been  given.  The  duration  is  nob  known  definitely.  From 
the  rich  experience  of  the  British  army  in  India  Colonel  Firth 1  concludes 
that  immunity  begins  to  decline  in  about  two  and  one-half  years  after 
inoculation.  However,  even  after  four  and  five  years  the  typhoid  rate 
among  the  inoculated  is,  estimated  roughly,  one-fourth  that  of  unpro- 
tected troops. 

Sensitized  Typhoid  Vaccine  of  Gay  and  Claypool. — Gay  and  Clay- 
pool,2  who  have  made  an  extended  and  extensive  study  of  typhoid  im- 
munization, have  advocated  a  polyvalent,  sensitized  typhoid  vaccine 
sediment  for  prophylactic  immunization  against  typhoid  fever,  as  being 
superior  to  other  forms  of  typhoid  vaccine.  Force3  has  found  that  this 
vaccine  produces  less  reaction  and  Sawyer4  has  found  it  more  protective 
than  several  other  types  of  commercial  vaccine.  Gay's  vaccine  consists, 
of  the  ground  sediment  of  a  mixed  polyvalent  vaccine  that  has  been 
sensitized  by  an  antityphoid  serum  and  then  killed  and  precipitated 
by  alcohol.  From  this  ground  culture  the  endotoxins  are  extracted 
by  carbolated  saline  solution  and  the  remaining  sediment  of  bacterial 
bodies  alone  used  for  prophylaxis  and  in  the  treatment  of  typhoid  fever. 

For  prophylactic  purposes  TTF  mg.  of  dried  bacteria,  which  corresponds 

1  Jour.  Roy.  Army  Med.  Corps,  1911,  xvi,  589. 

2  Archiv.  Int.  Med.,  1913,  xii,  613;  1913,  xii,  622;  1914,  xiii,  471;  1914,  xiv,  602- 
1914,  xiv,  662,  669,  and  671;  1916,  xvii,  303. 

3  Amer.  Jour.  Pub.  Health,  1913,  iii,  750. 

4  Jour.  Amer  Med.  Assoc.,  1915,  Ixv,  1413. 


692  ACTIVE    IMMUNIZATION 

to  an  original  bacteria  count  of  about  750,000,000,  is  administered  by 
subcutaneous  injection  every  other  day  until  three  or  four  doses  have 
been  given.  This  vaccine  is  claimed  to  produce  a  better  and  more  prompt 
immunity  response  than  other  vaccines.  It  has  also  been  used  in  the 
treatment  of  typhoid  fever  by  intravenous  injection. 

Results. — The  value  of  the  typhoid  prophylactic  therapy  is  best 
shown  in  the  army,  where  conditions  are  better  controlled  than  is  possible 
in  civilian  life.  In  1911,  of  a  division  of  United  States  troops,  about 
20,000  men  along  the  southern  boundary,  only  two  cases  of  typhoid 
fever  developed  and  both  recovered.  During  this  same  period  of  time 
49  cases  were  reported  in  the  city  of  San  Antonio,  with  19  deaths.  The 
soldiers  mixed  freely  in  the  city,  ate  of  fruits  and  vegetables,  drank  of 
the  same  water,  and  in  this  manner  were  freely  exposed,  although  the 
sanitary  conditions  in  the  camp  were  excellent. 

In  1898,  during  the  Spanish  War,  there  were  assembled  at  Jackson- 
ville, Florida,  10,759  troops,  among  whom  there  were  certainly  1729 
cases  of  typhoid,  and  including  the  suspected  cases,  this  figure  reached 
2693  cases,  with  248  deaths.  This  camp  continued  about  as  long  as 
that  in  1911,  the  climatic  conditions  and  water  supplies  being  practically 
the  same,  but  the  sanitary  conditions  were  bad.  The  remarkable  dif- 
ference in  the  typhoid  rate  cannot,  however,  be  reasonably  explained  by 
perfect  camp  sanitation,  and  the  results  in  1911  leave  no  doubt  as  to  the 
value  of  antityphoid  vaccination. 

Excellent  results  have  been  reported  by  Spooner,  Hachtel  and  Stoner, 
and  others  as  to  the  prophylactic  value  of  typhoid  immunization  in 
hospital-training  schools  for  nurses,  insane  asylums,  and  other  public 
institutions. 

Recommendations. — In  view  of  the  satisfactory  results  obtained  in 
the  army,  typhoid  vaccination  is  now  obligatory  on  all  members  of  the 
army  and  navy  corps.  Protection  of  the  individual  by  immunization 
is  the  only  measure  of  protection  independent  of  surroundings  and 
effective  under  all  conditions. 

Typhoid  inoculation  in  civilian  practice  cannot  be  as  wide-spread 
or  as  readily  performed  as  vaccination  against  smallpox,  as  the  prophy- 
lactic must  be  administered  subcutaneously  and  more  than  one  dose  is 
necessary. 

1.  Our  various  State  and  city  boards  of  health  should  endeavor  to 
educate  the  laity,  and,  if  necessary,  offer  the  prophylactic  free  of  charge 
in  order  to  build  up  a  vaccinated  community  as  far  as  this  is  possible. 

2.  Persons  coming  in  intimate  contact  with  typhoid  patients,  such 


PROPHYLACTIC    IMMUNIZATION   OR   VACCINATION  693 

as  physicians,  nurses,  and  attendants  in  hospitals,  should  be  immunized. 
Hospital  authorities  are  justified  in  making  typhoid  vaccination  ob- 
ligatory on  all  applicants  for  admission  to  training  schools. 

3.  All  inmates  of  asylums,  homes,  and  other  public  institutions 
under  forty-five  years  of  age  should  be  immunized  and  the  State  should 
be  ready,  if  necessary,  to  furnish  the  vaccine. 

4.  The  physician  and  nurse  should  urge  vaccination  upon  all  the 
members  of  a  family  when  there  is  a  typhoid  patient  among  them. 

5.  The  physician  should  especially  advise  immunization  of  those 
about  to  leave  their  homes  for  a  vacation  in  some  neighboring  seashore 
or  mountain  resort. 

6.  In  times  of  epidemics  of  typhoid  fever  the  physician  should  urge 
vaccination  of  all  over  whom  he  has  influence.     Thorough  vaccination 
with  proper  sanitary  conditions  offers  the  best  hope  of  eradicating  this 
dreaded  disease. 

PARATYPHOID  FEVER 

According  to  recent  experiences  in  the  European  armies  prophy- 
lactic immunization  with  typhoid  vaccine  does  not  afford  protection 
against  infections  with  Badllis  paratyphosus  A  and  Badllis  paraty- 
phosus  B.  In  this  country  various  investigators  have  estimated  that 
about  2  to  4  per  cent,  of  the  cases  of  so-called  " clinical  typhoid  fever" 
are  due  to  paratyphoid  infections;  apparently  the  majority  of  these  are 
with  Badllis  paratyphosus  B.  It  would  appear  advisable  therefore  to 
immunize  with  these  microorganisms  in  addition  to  Bacillis  typhosus, 
and  particularly  members  of  the  Army,  Navy,  National  Guards,  and  all 
volunteer  organizations  called  into  service  in  time  of  war.  For  over  a 
year  I  have  been  using  routinely  a  mixed  vaccine  prepared  in  the  same 
manner  as  the  typhoid  vaccine,  each  cubic  centimeter  of  which  contains 
500,000,000  typhoid  bacilli  of  the  army  strain  and  250,000,000  each  of 
Bacillis  paratyphosus  A  and  B,  the  latter  strains  being  selected  from  a 
number  on  the  basis  of  stimulating  the  production  of  agglutinins  to  a 
well-marked  degree  in  persons  and  rabbits.  The  first  dose  of  this  mixed 
vaccine  is  \  c.c. ;  three  more  injections  are  made  at  intervals  of  a  week  of 
1  c.c.  each,  making  a  total  of  four  injections.  In  the  Army  a  mixed 
vaccine  of  paratyphoid  bacilli  is  generally  administered  after  the  usual 
course  of  three  injections  of  typhoid  vaccine,  and  in  the  same  manner. 
Three  doses  are  given;  the  first  dose  contains  500,000,000  Badllus  para- 
typhosus A  and  300,000,000  Badllus  paratyphosus  B;  the  second  and 
third  doses  are  the  same,  and  contain  1,000,000,000  Badllus  paraty* 
phosus  A  and  600,000,000  Badllus  paratyphosus  B. 


694  ACTIVE    IMMUNIZATION 

TYPHUS  FEVER 

Preliminary  studies  upon  prophylactic  immunization  of  persons  with 
Bacillus  typhi  exanthematici,  a  Gram-positive  pleomorphic  bacillus 
secured  in  blood-cultures  from  persons  suffering  with  typhus  fever  by 
Plotz,  Olitslsy  and  Baehr,1  seem  to  indicate  that  a  vaccine  of  this  bacillus 
is  capable  of  reducing  the  incidence  of  the  disease,  although  it  does  not 
produce  an  absolute  immunity  to  typhus  fever  (Plotz,  Olitsky,  and 
Baehr).2  The  vaccine  is  polyvalent  and  is  sterilized  by  heating  to  58° 
to  60°  C.  for  from  half  an  hour  to  one  hour.  The  suspension  is  so  diluted 
that  each  cubic  centimeter  contains  about  2,000,000,000  bacteria,  and 
is  preserved  with  0.5  per  cent,  phenol  or  tricresol.  Three  injections  con- 
sisting of  0.5,  1,  and  1  c.c.,  respectively,  have  been  given  in  five-  or  six- 
day  intervals.  The  subject  is  in  the  experimental  stage. 

PNEUMONIA 

In  the  prophylaxis  of  lobar  pneumonia  by  active  immunization 
Wright3  has  had  an  unusually  rich  experience  in  the  Rand  mining 
district  of  South  Africa  where  a  severe  type  of  pneumococcus  pneu- 
monia has  claimed  a  high  morbidity  and  mortality  among  the  natives. 
After  a  considerable  amount  of  experimentation  the  administration  of 
a  single  large  dose  of  stock  vaccine  containing  1,000,000,000  cocci  was 
generally  employed.  Wright  thinks  that  this  vaccine  reduced  the  inci- 
dence of  pneumonia  among  the  natives  during  the  first  three  months 
following  inoculation.  Later  reports  of  this  work  have  failed  to  establish 
the  efficacy  of  active  immunization  in  preventing  the  development  of 
pneumonia  or  materially  lowering  the  mortality.  Since,  however,  re- 
cent investigations  by  Lister4  have  shown  the  existence  of  different 
serologic  types  of  pneumococci  on  the  Rand,  an  improvement  in  the 
efficiency  of  Wright's  vaccine  may  have  been  made  by  using  a  mixed 
vaccine  of  these  special  strains;  if  a  vaccine  is  employed  as  a  prophy- 
lactic measure  in  pneumonia,  it  should  be  prepared  of  a  number  of 
strains  belonging  to  types  I  and  II  at  least  and  several  injections  should 
be  given  at  intervals  of  five  or  seven  days.  The  protection  is  likely  to  be 
of  short  duration,  but  the  method  would  appear  worthy  of  trial  under 
these  conditions  and  particularly  during  the  seasons  when  pneumonia 

is  prevalent. 

PLAGUE 

In  view  of  the  frightful  infectiousness  and  mortality  of  plague, 
prophylactic  measures  are  highly  desirable.  Extermination  of  rats  and 

1  Jour.  Infect.  Dis.,  1915,  xvii,  1.        2  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  1597. 

3  Drugs  and  Vaccines  in  Pneumonia,  1915,  Paul  B.  Hoeber,  New  York. 

4  South  African  Inst.  for  Med.  Res.,  Johannesberg,  Dec.  22,  1913. 


PROPHYLACTIC   IMMUNIZATION   OR   VACCINATION  695 

ground  squirrels,  especially  of  the  former,  about  the  wharves  of  seaport 
cities  arid  towns  is  highly  essential,  as  the  disease  is  transmitted  by  the 
fleas  of  these  rodents.  Aside  from  sanitary  measures,  plague  vaccine, 
especially  that  of  Haffkine,  has  now  been  used  extensively,  with  encour- 
aging results. 

Preparation  of  Plague  Vaccine. — The  Haffkine  prophylactic  is  pre- 
pared by  growing  pure  cultures  of  Bacillus  pestis  in  flasks  of  neutral 
bouillon  to  which  a  few  drops  of  sterile  olive  oil  or  butter-fat  have  been 
added,  to  serve  as  floats  from  which  the  surface  growth  of  the  bacilli 
can  take  place.  The  flasks  are  cultivated  at  from  25°  to  30°  C.  for  five 
to  six  weeks,  and  are  shaken  every  two  or  three  days,  by  which  the  hang- 
ing, stalactite-like  colonies  are  thrown  down,  so  that  a  new  crop  of  the 
bacilli  can  develop  in  contact  with  the  air. 

After  growing  for  six  weeks  the  purity  of  the  culture  in  each  flask  is 
tested  by  subcultures  on  agar  and  by  direct  smears.  The  masses  of 
bacilli  are  broken  up  by  shaking,  and  the  material  is  sterilized  by  heating 
at  65°  C.  for  from  one  to  three  hours.  Phenol  is  added  to  the  point  of 
0.5  per  cent.,  and  the  fluid  is  tested  for  sterility  by  culture.  If  it  is 
found  sterile,  it  is  finally  poured  into  small  vials  of  from  10  to  30  c.c. 
capacity. 

Kolle  prepares  a  vaccine  by  cultivating  the  bacillus  for  two  days  in 
flasks  of  agar  measuring  10  by  9.5  cm.  Each  surface  of  agar  equals 
about  15  ordinary  agar  slant  cultures,  and  an  agar  slant  holds  about 
15  loopsful  of  culture  (4  mm.  loop).  A  loop  of  this  size  holds  about  2 
mg.  of  organisms,  and,  accordingly,  a  flask  of  agar  contains  about  225 
loopsful  of  culture,  or  225  doses  of  2  mg.  each.  Kolle  prepares  the 
vaccine  in  amounts  of  0.5  c.c.  per  dose  (2  mg.  of  bacilli),  and  the  growths 
in  each  flask  are  removed  with  112.5  c.c.  of  sterile  normal  salt  solution. 
The  emulsion  is  shaken  to  break  up  clumps,  heated  for  one  hour  at  70° 
C.,  and  tested  for  sterility.  It  is  then  preserved  with  phenol  or  tricresol 
and  placed  in  ampules  containing  0.5  c.c.  each. 

The  German  Plague  Commission  strongly  recommended  the  use  of 
twenty-four-  to  forty-eight-hour-old  agar  cultures  instead  of  the  old 
bouillon  cultures  employed  in  the  preparation  of  Haffkine's  vaccine. 

Kolle  and  Strong  have  also  employed  living  cultures  of  greatly  re- 
duced virulence  for  the  immunization  of  man. 

Lustig  and  Galeotti  prepare  a  vaccine  of  the  toxic  precipitate  pro- 
duced by  dissolving  the  bacilli  in  a  1  per  cent,  solution  of  caustic  soda 
and  neutralizing  with  1  per  cent,  of  acetic  acid.  This  precipitate  is 
dried  in  vacuo  and  redissolved  in  a  weak  solution  of  sodium  bicarbonate, 
the  dose  for  adults  being  0.0133  gm.  of  solid  substance. 


696  ACTIVE   IMMUNIZATION 

Terni  and  Bandi  inoculate  rabbits  or  guinea-pigs  intraperitoneally 
with  the  bacillus,  and  just  preceding  or  directly  after  death  they  collect 
the  peritoneal  exudate,  in  which  the  organisms  are  allowed  to  continue 
growing  for  twelve  hours  more.  The  bacilli  are  then  killed  at  a  low 
temperature,  and  the  fluid  thus  obtained,  after  a  preservative  has  been 
added,  constitutes  the  vaccine. 

Dosage. — The  ordinary  dose  of  Haffkine's  prophylactic  for  adult 
males  is  from  3  to  3.5  c.c. ;  for  adult  females,  from  2  to  2.5  c.c.  Haffkine 
himself  has  injected  larger  quantities  without  resulting  harm.  He 
recommends  giving  a  second  injection  after  from  eight  to  ten  days. 
The  injections  are  given  subcutaneously,  with  a  sterile  syringe,  into  the 
upper  arm  or  elsewhere  in  areas  where  the  skin  is  not  tightly  bound  down. 

The  local  and  constitutional  effects  are  similar  to  those  in  typhoid 
except  that  they  are  slightly  intensified.  The  inoculation  is  followed 
by  redness  and  swelling  at  the  seat  of  inoculation,  and  general  symp- 
toms in  the  form  of  rise  of  temperature  and  a  feeling  of  illness.  The 
latter  pass  off  in  twenty-four  hours,  but  the  patient  should  rest  during 
the  first  day  after  inoculation. 

Duration  of  Immunity. — The  immunity  is  apparent  a  few  days  after 
inoculation,  but  is  of  short  duration.  In  India  the  protection  is  believed 
to  last  at  least  three  months  and  possibly  longer.  In  times  of  epidemic 
the  inoculations  should  be  repeated  at  least  two  or  three  times  a  year. 
The  brief  duration  of  the  immunity  is  probably  one  reason  why  better 
results  are  not  secured.  Best  results  are  observed  during  epidemics  when 
protection  is  afforded  for  a  short  time,  or  until  the  danger  is  past.  In 
countries  or  localities  where  the  disease  is  endemic,  persons  may  refuse 
repeated  inoculation  and  thus  become  susceptible  to  infection. 

Results. — In  the  pneumonic  variety  of  plague  the  prophylactic  does 
little  or  no  good,  a  finding  that  has  also  been  shown  experimentally  by 
Strong  and  Teague.1 

In  the  bubonic  variety  Haffkine's  vaccine  has  in  general  yielded 
encouraging  results.  The  protection  is  not  absolute;  the  immunity 
is  of  relatively  short  duration,  and  therefore  good  results  are  not  so 
readily  appreciated  when  the  disease  is  endemic.  The  mortality  among 
the  inoculated  is  much  lower,  i.  e.,  11  to  41  per  cent.,  as  compared  with 
50  to  92  per  cent,  among  the  non-immunized.  Haffkine  summarized 
his  results  a  few  years  ago  as  follows:  Among  186,797  inoculated 
persons  there  were  3999  attacks,  or  1.8  per  cent.;  among  639,630  un- 
inoculated  persons  there  were  49,433  attacks,  or  7.7  per  cent.,  with  29,733, 
or  4.7  per  cent,  of  deaths. 

1  Philippine  Jour.  Sci.,  1913,  vii. 


PROPHYLACTIC    IMMUNIZATION   OR   VACCINATION  697 

The  Indian  Plague  Commission  a  few  years  ago  reported  as  follows : 

1.  Inoculation  sensibly  diminishes  the  incidence  of  attacks  of  plague. 
It  is,  however,  not  an  absolute  protection  against  the  disease. 

2.  The  death-rate  is  markedly  diminished  by  its  means,  not  only 
the  incidence  of  the  disease,  but  also  the  fatality,  being  reduced. 

3.  The  protection  is  not  conferred  on  those  inoculated  for  the  first 
few  days  after  the  injection. 

4.  The  duration  of  the  immunity  is  uncertain,  but  it  seems  to  last 
for  a  number  of  weeks,  if  not  for  months. 

After  the  disease  has  once  developed,  vaccination  is  of  no  avail. 
When  there  is  eminent  danger  of  infection,  vaccine  and  antiserum  should 
be  given  together. 

CHOLERA 

Protective  inoculation  against  cholera  was  first  practised  by  Ferran, 
a  Spaniard,  in  1884,  although  little  definite  knowledge  as  to  the  value 
of  the  procedure  resulted  from  his  work.  He  is  said  to  have  used  impure 
cultures  of  bacilli  isolated  from  the  feces  of  cholera  patients.  Broth 
cultures  were  prepared,  and  the  living  organisms  injected  subcutane- 
ously,  using  eight  drops  for  the  first  and  0.5  c.c.  for  the  second  and  third 
doses,  the  injections  being  given  at  intervals  of  six  or  eight  days.  While 
his  method  and  results  have  been  questioned,  he  was,  however,  the 
first  to  use  a  method  employed  later,  with  some  modifications,  by  Haff- 
kine  in  India  with  good  results. 

Preparation  of  Cholera  Vaccine.— Haffkine,  following  Pasteur's 
method  with  anthrax,  uses  two  vaccines, — a  weaker  and  a  stronger, — 
living  microorganisms  being  used  in  both  and  injected  subcutaneously. 
Vaccine  No.  1  is  weaker,  and  is  obtained  by  growing  the  bacilli  on  agar 
at  a  temperature  of  39°  C.  Vaccine  No.  2  is  composed  of  more  virulent 
organisms,  prepared  by  passing  the  vibrios  through  a  series  of  guinea- 
pigs  until  a  strain  is  obtained  which  is  invariably  fatal  to  these  animals 
within  twelve,  or  at  least  twenty-four,  hours.  Cultures  are  grown  on 
agar,  washed  off  with  8  c.c.  of  sterile  bouillon  or  saline  solution,  and 
administered  in  doses  of  1  c.c.,  which  is  equivalent  to  about  two  loopsful 
(4  mm.)  or  4  mg.  of  living  bacilli. 

Kolle  has  shown,  however,  both  by  animal  experimentation  and  in 
the  human  being,  that  heat-killed  cultures  are  equally  good,  and  that 
living  cultures  are  unnecessary  and  may  be  undesirable. 

By  this  method  the  vaccine  is  prepared  by  cultivating  a  virulent 
strain  on  flasks  of  agar  for  twenty-four  hours,  as  described  in  the  pre- 


698  ACTIVE   IMMUNIZATION 

paration  of  plague  vaccine,  and  removing  the  growths  with  sufficient 
salt  solution  so  that  1  c.c.  shall  contain  one  loopful  (4  mm.)  of  organisms 
(2  mg.).  The  emulsion  is  then  shaken  to  break  up  clumps,  heated  to 
60°  C.  for  from  one-half  to  one  hour,  cultured  to  determine  sterility,  and 
preserved  with  0.5  per  cent,  phenol. 

Strong  has  proposed  the  use  of  the  products  obtained  by  "autolytic 
digestion"  of  the  organism,  i.  e.}  by  incubating  an  emulsion  of  them  in 
sterile  water,  in  which  they  break  up  spontaneously.  Twenty-four-hour 
agar  cultures  are  removed  with  sterile  water,  placed  in  a  sterile  flask, 
and  kept  at  a  temperature  of  60°  C.  for  twenty-four  hours.  The  mixture 
is  then  put  aside  in  the  incubator  for  from  two  to  five  days.  The  best 
results  are  obtained  apparently  after  five  days'  autolytic  digestion. 
After  such  digestion  the  emulsion  is  filtered  through  a  Reichel  filter. 
The  fluid  thus  obtained  must,  of  course,  be  examined  for  sterility  and 
carefully  standardized  before  being  used  as  a  human  vaccine. 

Dosage. — Kolle's  vaccine  is  given  subcutaneously  in  two  injections 
about  a  week  apart — 1  c.c.  the  first  time  and  2  c.c.  the  second  time. 

Haffkine's  vaccines  are  given  in  the  same  manner  at  an  interval  of 
five  days. 

The  local  effects  are  usually  marked  by  more  or  less  pain  and  edema, 
which  subside  in  forty-eight  hours.  The  constitutional  effects  are  not 
infrequently  severe,  being  marked  by  malaise,  fever  (100°— 101°  F.), 
nausea  and  vomiting,  followed  the  next  day  in  about  10  per  cent,  of 
persons  by  transient  diarrhea.  Usually  all  symptoms  have  disappeared 
within  seventy-two  hours. 

Results. — Haffkine's  prophylactic  vaccine  has  yielded  favorable 
results  in  India.  Powell  reports  198  cases  of  cholera  among  6549  non- 
immunized  persons,  with  a  total  mortality  of  124.  Of  5778  inoculated 
persons,  there  were  27  cases,  with  14  deaths.  Much  better  results  were 
obtained  with  Kolle's  vaccine,  and  it  is  now  generally  used  in  preference 
to  the  Haffkine  vaccines. 

Murata,  during  an  epidemic  in  Japan  in  1902,  vaccinated  77,907 
persons.  Of  these,  47,  or  0.06  per  cent.,  developed  cholera,  and  20, 
or  0.02  per  cent.,  died.  Of  825,287  uninoculated  persons,  1152,  or  0.13 
per  cent.,  died.  During  a  recent  epidemic  in  Russia  Franschetti  inocu- 
lated 11,178  persons.  Of  these,  8  contracted  cholera  and  1  died.  In 
St.  Petersburg,  during  1907-08,  30,000  persons  were  inoculated.  Of 
these,  12  developed  cholera  and  4  died.  Of  10,000  uninoculated  per- 
sons, 68  contracted  the  disease. 

It  appears  justifiable,  therefore,  to  conclude  that  inoculation  confers 


PROPHYLACTIC    IMMUNIZATION    OR    VACCINATION 


699 


some  immunity.  This  protection  may  be  apparent  after  the  first  dose, 
but  is  more  marked  after  the  second.  The  immunity  conferred  is  far 
from  being  absolute,  and  it  is  noteworthy  that  while  the  prophylactic 
diminishes  the  liability  of  the  inoculated  person  to  cholera,  it  has  less 
influence  on  the  mortality  when  the  disease  occurs  in  those  who  have 
been  vaccinated. 

Used  in  conjunction  with  modern  sanitary  regulations,  however, 
Kolle's  vaccine  certainly  proves  of  value  and  should  be  used  in  combating 
epidemics. 

OTHER  DISEASES 

.  Dysentery. — Protective  vaccination  against  bacillary  dysentery  has 
been  attempted,  but  has  not  as  yet  yielded  satisfactory  results.  Shiga 
practised  mixed  active  and  passive  immunization  (vaccine  plus  immune 
serum)  on  10,000  persons,  and  while  this  did  not  decrease  the  number 
of  infections,  a  lower  mortality  resulted.  The  various  types  of  the 
dysentery  bacillus  and  the  high  toxicity  of  the  vaccines  are  obstacles 
to  the  more  general  use  of  protective  inoculation  in  this  condition. 

Cerebrospinal  Meningitis. — Sophian  and  Black1  have  shown  ex- 
perimentally that  a  polyvalent  meningococcic  vaccine,  heated  to  50°  C., 
standardized  in  the  usual  manner,  and  given  in  three  injections,  in 
doses  of  100,000,000,  500,000,000,  and  1,000,000,000,  at  intervals  of  a 
week,  appears  to  afford  a  high  degree  of  protection.  In  the  blood- 
serums  of  inoculated  persons  these  observers  were  able  to  demonstrate 
opsonins,  agglutinins,  and  complement-fixing  amboceptors.  All  evi- 
dence points  to  the  efficacy  of  prophylactic  vaccination,  as  only  a 
moderate  degree  of  immunity  may  give  complete  protection  against  the 
disease.  The  method  has  not  thus  far  received  extensive  trial,  but  in 
the  presence  of  an  epidemic  its  harmlessness  and  apparent  value  should 
be  borne  in  mind. 

Scarlet  Fever. — Several  Russian  physicians,  particularly  Gabrick- 
evski,2  Longovi,  Nitikin,  Shamarin,  and  others,  have  secured  good 
results  from  a  method  of  prophylactic  vaccination  against  scarlet  fever 
with  a  polyvalent  vaccine  of  scarlet-fever  streptococci.  Heat-killed 
vaccines  were  given  in  three  successive  doses.  In  this  country  the 
method  has  been  tried  by  Kolmer,3  who  found  that  while  inoculations 
with  a  heat-killed  streptococcic  vaccine  cannot  prevent  scarlet  fever 

1  Jour.  Amer.  Med.  Assoc.,  1912,  lix,  527.     Black,  ibid.,  1913,  Ix,  1289. 

2  Russk.  Vratch,  St.  Petersburg,  1906,  x,  469. 

3  Penna.  Med.  Jour.,  February,  1912. 


700  ACTIVE   IMMUNIZATION 

itself,  such  inoculations  may,  however,  prevent  a  severe  attack  of  the 
disease  by  producing  some  immunity  against  secondary  streptococcic 
infections. 

Pertussis. — Several  observers  have  advocated  prophylactic  immuni- 
zation against  pertussis  among  children  who  are  exposed  or  likely  to  be 
exposed  by  the  subcutaneous  administration  of  a  stock  pertussis  vaccine 
in  dose  of  about  100,000,000  bacilli  at  intervals  of  a  week. 

PROTECTIVE  IMMUNIZATION  AMONG  THE  LOWER  ANIMALS 

Since  discoveries  in  bacteriology  and  immunity  have  usually  been 
intimately  associated  with  animal  experimentation,  it  is  not  strange 
that  the  lower  animals  should  have  been  the  first  to  benefit  from  the 
knowledge  thus  gained.  As  a  consequence,  vaccine  therapy,  both 
prophylactic  and  therapeutic,  is  being  extensively  used  in  veterinary 
practice  with  good  results. 

ANTHRAX 

This  was  one  of  the  first  vaccines  studied  by  Pasteur,  and  as  a  prophy- 
lactic measure,  it  has  proved  of  great  value.  It  finds  its  greatest  field 
of  usefulness  in  case  of  an  outbreak  of  anthrax,  when  it  is  used  to  pro- 
tect the  uninfected  members  of  a  herd,  as  well  as  any  animals  pasturing 
on  infected  areas. 

In  preparing  the  vaccine  Pasteur  was  hampered  by  the  fact  that  the 
spores  of  anthrax  bacilli  retain  the  virulence  of  the  original  bacilli.  As 
the  result  of  extended  experiments,  however,  he  discovered  a  means  of 
attenuating  the  virulence  of  cultures  by  growing  the  bacilli  at  a  tem- 
perature of  42°  C.;  he  also  found  that  inoculation  with  these  attenuated 
bacilli  would  effectively  vaccinate  sheep  and  cattle,  and  so  protect  them 
against  an  attack  of  the  disease. 

One  of  the  most  dramatic  stories1  in  the  history  of  science  is  the 
account  of  the  method  by  which  Pasteur  demonstrated  his  discovery  to 
the  public.  Certain  harsh  critics,  having  heard  of  Pasteur's  ability  to 
prevent  anthrax  in  laboratory  experiments,  and  anxious  to  humiliate 
him,  sent  him  a  public  challenge  to  demonstrate  the  experiment  on  a 
practical  scale  at  a  farm  in  the  country.  A  number  of  farmers  offered 
to  place  60  sheep  at  his  disposal.  The  challenge  was  immediately 
accepted,  and  Pasteur  mapped  out  a  plan  of  action,  in  which  he  safe- 
guarded himself  by  making  no  half-statements,  but  boldly  promised 
complete  success.  Of  the  total  number,  25  sheep  were  to  be  vaccinated 
and  25  were  to  remain  unvaccinated.  A  fortnight  later  all  50  were  to 
1  Narrated  by  Elizabeth  Fraser. 


PROTECTIVE    IMMUNIZATION   AMONG   THE    LOWER   ANIMALS        701 

receive  a  lethal  dose  of  the  fully  virulent  anthrax.  He  declared  that 
the  25  non-vaccinated  sheep  would  die,  whereas  the  25  vaccinated  would 
remain  alive  and  well.  The  remaining  10  sheep  were  to  serve  as  controls. 

The  challenge  and  its  acceptance  were  widely  advertised  in  the 
journals,  and  Pasteur  was  made  the  subject  for  many  witticisms.  Ex- 
citement ran  high,  and  a  large  crowd,  comprised  of  physicians,  veterin- 
ary surgeons,  journalists,  farmers,  etc.,  accompanied  Pasteur  to  the 
farm  (Pouilly  le  Fort)  where  he  was  to  make  the  final  test  by  inoculating 
the  deadly  anthrax.  One  vaccinated  animal  developed  a  temperature 
overnight,  a  fact  that  caused  Pasteur  much  anxiety.  On  going  to  the 
farm  the  next  day,  however,  again  followed  by  the  crowd,  he  found  all 
the  vaccinated  animals  well!  Of  the  unvaccinated,  22  were  dead,  and 
the  others  died  during  the  following  night.  Pasteur's  triumph  was 
complete,  and  the  possibility  of  preventive  vaccination  was  demonstrated 
to  the  world. 

The  vaccine  is  prepared  of  attenuated  cultures  of  virulent  anthrax 
bacilli. 

Vaccine  No.  1  is  weakest  or  lowest  in  virulence,  and  the  first  to  be 
injected.  This  vaccine  is  prepared  by  growing  virulent  anthrax  bacilli 
at  a  temperature  of  42°  C.  for  from  six  to  ten  weeks,  or  until  tV  loopful 
of  the  culture,  when  injected  into  rabbits,  guinea-pigs,  and  mice,  will 
show  virulence  for  mice  only,  but  not  for  guinea-pigs  and  rabbits. 

Vaccine  No.  2  is  prepared  by  growing  virulent  anthrax  bacilli  at 
42°  C.  for  about  twenty  days,  or  until  tV  loopful  is  virulent  for  mice, 
partly  so  for  guinea-pigs,  and  not  at  all  for  rabbits. 

Vaccine  No.  3  is  not  generally  used  except  for  immunizing  sheep  and 
goats.  When,  however,  it  is  required,  it  is  made  as  follows:  Virulent 
anthrax  bacilli  are  grown  at  42°  C.  until  yV  loopful,  when  injected  into 
mice,  guinea-pigs,  and  rabbits,  will  be  virulent  for  all  the  mice,  all  the 
guinea-pigs,  and  some  of  the  rabbits. 

The  vaccines  are  prepared  in  ampules  containing  1  c.c.  of  the  emul- 
sion, each  representing  one  dose,  to  be  injected  subcutaneously.  Vac- 
cine No.  2  is  injected  twelve  days  after  Vaccine  No.  1,  and  No.  3  after 
the  same  interval  following  Vaccine  No.  2.  The  resulting  immunity 
usually  lasts  about  six  months. 

In  instances  where  it  is  desirable  to  immunize  a  herd  before  turning 
them  out  to  pasture  on  infected  areas  it  is  well  to  inoculate  the  animals 
in  the  early  spring,  keeping  them  in  the  stable  during  the  time  required 
for  at  least  two  vaccinations,  for  the  reason  that,  immediately  after 
vaccination,  the  animals  may  become  hypersusceptible  to  infection. 


702  ACTIVE    IMMUNIZATION 

When  conducted  in  a  careful  manner  by  a  competent  veterinarian, 
anthrax  vaccination  has  proved  fairly  successful. 

BLACKLEG 

Blackleg  vaccine  is  used  entirely  as  a  prophylactic  agent,  for  the 
disease  runs  too  acute  a  course  for  the  vaccine  to  exert  any  therapeutic 
influence. 

The  vaccine  is  prepared  by  the  Bureau  of  Animal  Industry  in  the 
following  manner:  The  muscle  tissue  from  a  fresh  blackleg  tumor  is 
ground  in  a  mortar,  extracted  or  macerated  with  a  little  water,  and 
the  fluid  squeezed  through  cheese-cloth.  The  expressed  fluid  is  then 
evaporated  at  a  temperature  of  35°  C.  The  dry  brown  scale  is  run 
through  a  grinding  mill  and  heated  for  six  or  seven  hours  at  a  temperature 
of  from  94°  to  96°  C.  This  process  of  heating  attenuates  the  virulence 
of  the  bacilli  present  so  that,  when  injected,  they  produce  but  a  mild 
attack  of  the  disease.  The  Department  of  Agriculture  places  the  ground 
material  in  packages  containing  a  certain  number  of  doses.  These 
packages  are,  upon  request,  mailed  to  veterinarians,  who  dilute  the 
ground  muscle  with  as  many  cubic  centimeters  of  sterile  water  as  there 
are  doses  in  the  package.  One  cubic  centimeter  of  the  suspension  is 
injected  subcutaneously  in  some  convenient  area,  as,  e.  g.,  about  the 
shoulders. 

Blackleg  vaccine  should  be  applied  in  the  spring,  before  young  cattle 
are  turned  out  to  pasture  on  infected  areas. 

In  case  of  a  fresh  outbreak,  all  the  healthy  animals  in  the  herd  are 
to  be  vaccinated  as  soon  as  possible. 

Blackleg  vaccination  has  been  fairly  successful,  and  usually  confers 
an  immunity  lasting  for  a  period  of  about  six  months. 


ACTIVE   IMMUNIZATION   FOR   THERAPEUTIC   PURPOSES— BACTERIAL 

VACCINE  THERAPY 

Principles. — Although  bacterial  vaccines  have  been  extensively 
employed  in  the  treatment  of  various  diseases,  it  is  difficult  to  express 
an  opinion  as  to  their  real  value,  and  it  is  altogether  impossible  to  make 
dogmatic  statements  as  to  the  percentage  of  cases  in  which  they  will  be 
helpful  or  effect  a  cure,  or  as  to  what  result  may  be  expected  in  an  in- 
dividual case.  Following  Wright's  original  announcements,  this  special 
therapy  was  enthusiastically  received  by  the  profession,  and  in  a  short 
space  of  time  the  method  was  employed  experimentally  in  many  and 


ACTIVE    IMMUNIZATION   FOR   THERAPEUTIC   PURPOSES  703 

diverse  infections.     It  may  be  stated  that  in  many  infections  the  vac- 
cines may  be  beneficial,  but  they  should  be  used  only  under  proper 
conditions,  as  was  indicated  in  the  first  portion  of  this  chapter. 
It  may  be  stated  in  general  that: 

1.  Vaccine  therapy  has  a  special  field  of  usefulness  in  the  treatment 
of  chronic  infections. 

2.  Autogenous  vaccines  are  to  be  preferred  to  stock  vaccines,  and  in 
some  infections  the  former  must  be  used. 

3.  It  is  not  advisable  to  continue  the  use  of  the  same  vaccine  for 
more  than  several  doses  if  no  reaction  and  no  improvement  are  noted. 
New  cultures  should  be  made  to  determine  if  the  right  organism  is 
being  used,  or  if  reinfection  with  another  organism  has  occurred. 

4.  It  is  essential  that  the  vaccine  be  properly  prepared  and  not  over- 
heated. 

5.  It  is  highly  important  that  the  usual  forms  of  treatment  be  employed 
in  conjunction  with  the  vaccine  therapy.     Thus   abscesses   should  be 
incised;   proper  drainage  of  a  discharging  wound  afforded;   discharging 
ears  cleansed,  etc. 

6.  While  the  dose  should  not  be  too  large,  neither  should  it  be  too 
small,  nor  too  far  apart.     There  is  a  proper  dose  for  each  patient,  and 
this  may  be  determined  by  starting  with  a  small  dose  and  gradually 
increasing  it  until  some  reaction  is  secured.     An  efficient  dose  must 
necessarily  produce  some  reaction,  and    increased  doses    are  contra- 
indicated  so  long  as  any  sign  of  general  or  focal  reaction  is  produced  and 
so  long  as  steady  progress  is  maintained. 

INFECTED  WOUNDS 

In  the  treatment  of  infected  wounds  and  sinuses  autogenous  vac- 
cines are  frequently  of  considerable  value  in  those  infections  due 
to  staphylococci  and  streptococci.  In  chronic  suppurations  pseu- 
dodiphtheria  bacilli  and  Bacillus  pyocyaneus  are  commonly  found, 
but  these  microorganisms  are  likely  to  be  secondary  invaders.  While 
bacterial  vaccines  are  being  employed  the  usual  surgical  treatment  should 
be  given,  and  it  is  particularly  important  to  afford  adequate  drainage  and 
remove  foreign  bodies,  as  sutures  and  bits  of  necrotic  bone.  Great  care 
must  be  exercised  in  preparing  the  cultures  in  order  that  the  infecting 
bacteria  may  be  secured  for  the  vaccine.  In  wounds  likely  to  be  infected 
with  staphylococci  or  streptococci,  it  would  be  good  practice  to  admin- 
ister one  or  two  doses  of  a  stock  vaccine  in  an  effort  to  protect  against 
infection. 


704  ACTIVE  IMMUNIZATION 

FOCAL  INFECTIONS 

As  discussed  on  page  94,  recent  investigations  have  shown  that 
various  bacteria  and  particularly  strains  of  streptococci  may  gain 
entrance  to  the  blood  and  lymph  circulations  through  foci  of  infection, 
particularly  at  the  roots  of  teeth  and  in  the  tonsils.  Various  lesions 
and  particularly  arthritis  and  endocarditis  have  been  attributed  to 
this  mode  of  infection.  In  treatment  particular  attention  should  be 
given  to  the  detection  and  removal  of  these  foci,  and  autogenous  vaccines 
prepared  of  cultures  from  the  infected  tissues  may  be  of  aid. 

DISEASES  OF  THE  SKIN 

Furunculosis ;  Abscesses. — Furuncles  are  usually  caused  by  some 
member  of  the  group  of  staphylococci,  and  frequently  the  most  rational 
and  successful  form  of  therapy  is  by  means  of  bacterial  vaccines.  A 
stock  vaccine  of  Staphylococcus  aureus  may  prove  satisfactory,  and 
should  be  used  while  an  autogenous  vaccine  is  being  prepared.  For 
adults  the  initial  dose  may  be  100,000,000  cocci,  succeeding  doses 
gradually  increasing  until  1,000,000,000  are  given  at  one  time.  The 
injections  may  be  given  at  intervals  of  from  five  to  seven  days.  Follow- 
ing the  first  few  doses  a  slight  focal  and  some  constitutional  reaction 
should  be  secured.  After  all  the  lesions  have  disappeared,  one  or  two 
full  doses  at  intervals  of  several  months  will  continue  to  fortify  the 
patient  against  a  recurrence. 

Carbuncles. — These  are  invariably  caused  by  the  Staphylococcus 
aureus,  and  exceptionally  by  a  streptococcus.  The  urine  should  be 
examined  for  sugar,  and  even  if  the  patient  is  diabetic,  small  doses  of 
vaccine  may  be  of  value  when  used  in  conjunction  with  the  customary 
treatment. 

Sycosis. — Sycosis  vulgaris  is  usually  caused  by  the  Staphylococcus 
aureus  and  albus,  and  such  patients  are  frequently  very  rebellious  to  the 
ordinary  treatment.  An  autogenous  vaccine  is  very  helpful  in  some 
cases.  Due  care  should  be  exercised  in  making  cultures  to  secure  pus 
from  a  well-developed  lesion.  Relatively  large  doses  of  vaccine  are 
necessary,  and  treatment  is  usually  prolonged,  at  least  12  injections  being 
necessary  before  the  conclusion  is  reached  that  the  vaccines  are  of  no 
service.  As  a  rule,  the  condition  will  improve  under  vaccine  therapy, 
but  only  in  exceptional  cases  does  a  complete  cure  result. 

Acne. — Acne  is  frequently  caused  by  two  microorganisms — a  Staphy- 
lococcus and  the  acne  bacillus.  In  cases  showing  pustulation  a  Staphy- 
lococcus is  invariably  present,  and  exceptionally  the  bacillus  may  be 
found  alone  in  comedones.  Cultures  should  be  made  from  several 


ACTIVE    IMMUNIZATION    FOR   THERAPEUTIC    PURPOSES  705 

lesions,  being  careful  to  secure  pus  that  has  not  been  contaminated  by 
the  skin.  Stock  vaccines  may  .be  used,  although  autogenous  vaccines 
are  likely  to  yield  better  results.  In  view  of  the  frequency  of  intestinal 
disorders  among  persons  suffering  with  acne,  Strickler,  Schamberg,  and  I1 
have  studied  the  possible  influence  of  Bacillus  coli  communis  and  com- 
munior  by  means  of  complement-fixation  tests,  and  found  that  the  sera 
of  a  large  percentage  of  persons  suffering  with  acne  yielded  positive 
reactions.  On  the  basis  of  these  results  I  have  been  incorporating  in 
the  vaccine  one  or  more  strains  of  Bacillus  coli  isolated  from  the  patient's 
feces;  this  modification  has  appeared  to  increase  the  efficacy  of  the 
vaccine.  It  is  well  to  administer  a  mixed  vaccine  of  the  straphylococcus, 
acne  bacillus,  and  colon  bacillus,  especially  if  the  lesions  are  pustular. 
It  is  highly  essential  that  other  forms  of  treatment  be  instituted  while 
vaccines  are  being  tried,  as  the  treatment  is  usually  prolonged.  Ex- 
ceptionally, however,  a  brilliant  result  may  be  observed  after  a  few  injec- 
tions. I  generally  prepare  a  mixed  vaccine  of  10,000,000  acne  bacilli 
to  each  200,000,000  cocci  and  200,000,000  Bacillus  coli  per  1  c.c.  of 
vaccine.  The  first  few  doses  consist  of  0.5  c.c.,  and  later  this  amount 
may  be  increased  to  1  c.c.  per  dose.  Doses  are  given  every  five  to  seven 
days,  or  just  when  retrogression  is  observed  to  occur.  It  may  be  neces- 
sary to  use  large  doses,  and  in  any  case  the  treatment  is  prolonged  over 
many  weeks  and  months.  Most  cases  will  show  improvement,  but  few 
are  absolutely  cured  by  a  single  series  of  injections. 

Eczema. — The  prolonged  administration  of  an  autogenous  vaccine 
of  staphylococci  secured  from  the  scales  of  serous  exudate  of  eczema 
have  occasionaly  aided  in  the  treatment  of  obstinate  cases. 

Impetigo  and  Ecthyma. — The  administration  of  a  stock  vaccine  of 
Staphylococcus  albus  arid  streptococci  or,  preferably,  of  the  cocci  iso- 
lated from  the  pustules  of  impetigo  contagiosa  and  ecthyma  may  be  of 
considerable  value  in  the  treatment  of  infections  tending  to  pursue  a 
chronic  course.  Likewise  the  administration  of  an  autogenous  vaccine 
in  impetigo  herpetiformis  may  be  of  aid  in  the  treatment  of  this  fre- 
quently fatal  infection  in  puerperal  women. 

Dermatitis  Venenata. — The  most  common  forms  of  dermatitis  due 
to  poisonous  plants  are  those  caused  by  poison  ivy  or  oak  (Rhus 
toxicodendron) ,  and  of  sumach  or  dogwood  (Rhus  venenata).  Cer- 
tain persons  are  susceptible  to  a  poisonous  principle  of  these  plants 
and  develop  a  dermatitis  after  being  in  contact  with  them.  This  form 
of  dermatitis  is  characterized  by  urticarial  lesions  and  is  regarded  as  an 

1  Jour.  Cutan.  Dis.,  March,  1916. 
45 


706  ACTIVE    IMMUNIZATION 

anaphylactic  phenomenon  and  analogous  to  hay-fever.  Specific  treat- 
ment with  extracts  of  ivy,  sumac,  and  other  plants  aims  to  desensitize 
the  patient  or  produce  a  condition  of  anti-anaphylaxis.  Strickler, 
working  in  my  laboratory,  has  succeeded  in  the  treatment  of  a  number 
of  cases  of  severe  ivy-poisoning;  the  extract  is  injected  subcutaneously 
and  usually  affords  relief  within  twenty-four  hours.  As  in  hay-fever, 
the  desensitization  is  of  short  duration  and  the  injections  must  be  given 
at  short  intervals.  It  is  highly  probable  that  a  prolonged  series  of  in- 
jections will  effectively  and  permanently  desensitize. 

Ringworm. — Strickler1  has  succeeded  in  treating  ringworm  of  the 
scalp  with  a  polyvalent  vaccine  prepared  of  pure  cultures  of  the  ring- 
worm fungus.  Usually  seven  or  more  injections  were  necessary  at  in- 
tervals of  five  to  seven  days.  It  would  appear  that  this  form  of  therapy 
is  of  value  as  an  adjuvant  to  local  treatment  of  obstinate  infections. 

Erysipelas. — This  infection  is  usually  caused  by  the  streptococcus 
erysipelatis.  In  severe  cases  an  autogenous  vaccine  of  about  20,000,000 
cocci  per  cubic  centimeter  may  be  administered  every  three  or  four  days, 
and  frequently  aids  in  reducing  the  severity  of  the  inflammation  and 
overcoming  mental  unrest  and  physical  discomfort.  A  vaccine  may  be 
of  aid  in  the  treatment  of  subacute  and  chronic  types  of  this  disease. 
Stock  vaccines  are  of  little  or  no  value. 

GENITO-URINARY  DISEASES 

Cystitis. — Acute  or  subacute  cystitis  following  catheterization  after 
labor  or  surgical  operations  or  occurring  in  children  is  usually  caused  by 
a  member  of  the  group  of  colon  bacilli.  It  is  highly  essential,  in  order 
to  attain  success  with  vaccine  therapy,  that  urine  be  collected  aseptically 
and  the  causative  microorganism  secured  and  used  in  the  preparation 
of  a  vaccine,  as  stock  vaccines  are  of  little  or  no  value.  Exceptionally 
the  infection  may  be  due  to  another  microorganism,  either  alone  or  in 
conjunction  with  Bacillus  coli.  Treatment  with  an  autogenous  vaccine 
may  be  of  distinct  aid  in  lessening  the  symptoms  and  in  reducing  the 
amount  of  pus.  The  initial  adult  dose  of  Bacillus  coli  vaccine  should 
be  about  from  50,000,000  to  100,000,000  bacilli.  In  subacute  cystitis 
of  the  male,  due  to  stricture  of  the  urethra  or  enlarged  prostate,  or  in  the 
female,  due  to  perineal  injuries,  not  much  benefit  follows  its  use  until 
the  underlying  cause  is  corrected  or  removed. 

Pyelitis. — Pyelitis  in  children  and  adults  is  usually  due  to  Bacillus 
coli,  and  treatment  with  an  autogenous  bacterial  vaccine  has  frequently 
yielded  good  results.  The  urine  used  for  culture  must  be  obtained  under 
1  Jour.  Cutan.  Dis.,  March,  1915,  161. 


ACTIVE    IMMUNIZATION    FOR    THERAPEUTIC    PURPOSES  707 

aseptic  conditions  and  preferably  with  a  catheter.  Stock  vaccines  of  Ba- 
cillus coli  and  vaccines  prepared  of  Bacillus  coli  isolated  from  the  urine 
collected  under  ordinary  conditions,  and  therefore  likely  to  be  other 
than  that  strain  causing  the  infection,  are  of  little  value.  Children 
usually  bear  the  vaccine  very  well;  I  usually  administer  50,000,000  to 
100,000,000  bacilli  every  five  to  seven  days. 

Urethritis. — There  is  a  considerable  difference  of  opinion  as  regards 
the  efficacy  of  vaccines  in  the  treatment  of  acute  and  chronic  ure- 
thritis  of  gonorrhea!  origin.  A  polyvalent  stock  vaccine  of  gonococci  of 
proved  immunizing  powers  may  be  even  more  efficient  than  an  auto- 
genous one,  especially  if  the  latter  must  be  prepared  from  a  strain  that 
has  been  repeatedly  subcultured  in  order  to  obtain  the  vaccine  in  a  pure 
state,  or  from  one  that  has  lost  its  virulence  from  long  residence  in  the 
infected  urethra.  Owing,  therefore,  to  the  difficulty  of  isolating  and 
cultivating  gonococci,  stock  vaccines  have  been  generally  employed. 
In  subacute  urethritis  the  initial  dose  may  be  25,000,000;  if  complica- 
tions threaten,  less  than  this,  and  if  no  local  reaction  has  followed  more 
than  this,  is  given,  the  object  being  to  secure  a  slight  increase  of  the 
secretion,  which  should  become  more  purulent,  and  a  little  constitu- 
tional disturbance,  followed  by  lessening  of  local  pain  and  tenderness. 

In  chronic  urethritis  the  primary  infection  is  gonococcal,  although 
other  organisms,  such  as  the  Micrococcus  catarrhalis,  staphylococci,  and 
diphtheria  bacilli  may  have  some  relation  to  the  process.  A  stock 
gonococcal  vaccine  may  be  used  in  sufficient  dosage  to  evoke  a  reaction; 
not  infrequently,  however,  a  stock  and  an  autogenous  vaccine  or  vac- 
cines combined  yield  better  results.  Cultures  of  the  urethra  should 
be  made  only  after  thorough  cleansing  of  the  meatus  and  flushing  of  the 
urethra  with  sterile  salt  solution  and  massage  of  the  prostate,  cultures 
being  made  direct  from  the  prostatic  secretion  or  from  the  crypts  through 
the  urethroscope.  In  any  case  expert  local  treatment  should  be  given 
while  vaccines  are  being  used. 

Gonorrheal  Arthritis. — Polyvalent  stock  vaccines  of  gonococci  have 
generally  been  found  to  be  of  distinct  value  in  the  treatment  of  this 
troublesome  and  oftentimes  chronic  infection.  Local  treatment  of  the 
urethra  and  general  therapeutic  measures  should  be  employed.  It  may 
be  stated  that  vaccines  should  be  used  routinely  in  all  cases,  as  under 
any  condition  the  infection  is  likely  to  be  prolonged  and  tedious.  In  the 
acute  stages  the  doses  should  be  relatively  small,  about  10,000,000  cocci 
being  given  every  three  to  five  days.  In  the  subacute  stages  from 
50,000,000  to  100,000,000  may  be  given  at  intervals  of  from  five  to  ten 
days. 


708  ACTIVE    IMMUNIZATION 

Vulvovaginitis  of  Children. — Stock  goriococcal  vaccines  have  been 
used  quite  extensively  in  the  treatment  of  this  troublesome  infection. 
On  the  whole,  good  results  have  been  reported,  although  in  any  case 
final  judgment  must  be  reserved  until  thorough  bacteriologic  examina- 
tion shows  whether  the  tissues  are  really  free  from  infection  or  whether 
the  infection  has  subsided  and  become  chronic.  Smears  of  the  secretions 
alone  are  insufficient  to  determine  whether  a  cure  has  been  effected. 
Injecting  a  solution  of  1  :  2000  bichlorid  of  mercury  in  normal  saline 
solution  into  the  vagina,  followed  by  immediate  centrif ugalization  of  the 
washings  and  smears  of  the  sediment,  will  frequently  demonstrate  the 
presence  of  gonococci  that  will  not  otherwise  be  found.  If  vaccines  are 
used,  a  dose  of  from  5,000,000  to  10,000,000  every  five  to  seven  days 
may  be  employed. 

RESPIRATORY  DISEASES 

Rhinitis. — The  use  of  mixed  stock  vaccines  is  being  advocated  for 
prophylactic  immunization  against  recurrent  attacks  of  acute  rhinitis. 
Numerous  reports  by  Coates,  Fisher,  and  others  have  emphasized  the 
value  of  this  prophylactic  immunization.  With  Weston,  I  have  found 
autogenous  vaccines  of  some  value  in  lessening  the  severity  and  hasten- 
ing the  recovery  from  the  acute  rhinitis  of  scarlet  fever,  so  potent  a 
factor  in  the  dissemination  of  that  disease.  In  chronic  rhinitis  an  au- 
togenous vaccine,  prepared  by  growing  cultures  with  the  care  previously 
described,  may  be  of  distinct  value,  but  only  when  an  underlying  factor, 
such  as  a  malformation  or  adenoids,  has  been  removed,  and  only  when 
used  in  conjunction  with  efficient  local  treatment.  In  atrophic  rhinitis 
good  results  have  been  reported  by  various  observers  with  a  vaccine  of 
Bacillus  ozena  (Perez) ;  in  the  experience  of  Ersner,  working  in  my  labor- 
atory, the  results  have  been  usually  disappointing.  In  view  of  the  diffi- 
culty in  curing  this  disease,  a  vaccine  may  be  tried  as  an  adjuvant  to 
other  measures. 

Hay-fever. — Hay-fever  is  now  regarded  as  due  to  a  state  ol  anaphy- 
laxis  or  hypersensitiveness  to  the  pollen  of  certain  plants  and  grasses. 
Spring  fever  or  "rose  cold"  may  be  due  to  the  pollens  of  a  wide  variety 
of  grasses  and  plants  (red  top  timothy,  rye,  and  orchard  grass),  while 
autumnal  hay-fever  is  most  frequently  due  to  the  pollens  of  rag-weed 
and  golden-rod.  Since  the  brilliant  researches  of  Blackley,1  a  large  num- 
ber of  investigations  have  served  to  establish  the  nature  of  this  trouble- 
some disease  on  the  basis  of  idiosyncrasy  or  local  hypersensitiveness 
to  the  protein  of  one  or  more  plants.  According  to  Cooke  and  Vander 
1  Virchow's  Archives,  1877,  Ixx,  429;  Med.  Times  and  Gaz.,  1877,  2,  243. 


ACTIVE    IMMUNIZATION    FOR    THERAPEUTIC    PURPOSES  709 

Veer1  the  sensitization  is  not  directly  inherited,  although  the  tendency 
to  spontaneous  sensitization  is  inherited  as  a  dominant  character. 

Specific  treatment  with  pollen  extracts  aims  to  desensitize  the  patient. 
With  pure  pollens  at  hand  skin  tests  may  show  the  particular  one  to 
which  a  patient  is  hypersensitive.  A  small  abrasion  is  made  and  a 
minute  amount  of  the  powdered  pollen  applied;  the  development  of  an 
urticarial  lesion  within  ten  or  fifteen  minutes  indicates  that  the  person  is 
hypersusceptible  to  that  particular  pollen.  Ophthalmic  tests  have  also 
been  used  for  diagnosis  and  as  a  guide  in  treatment,  but  are  not  to  be 
recommended. 

The  satisfactory  preparation  of  hay-fever  vaccine  has  not  as  yet  been 
accomplished,  owing  to  the  tendency  of  preparations  to  deteriorate 
and  loose  in  antigenic  or  desensitizing  properties.  Hitchens  and  Brown2 
prepare  an  extract  of  the  pollen  grains  in  physiologic  salt  solution,  pre- 
cipitate with  acetone,  and  preserve  the  dried  precipitate  for  the  vaccine. 
For  use,  the  precipitate  is  dissolved  in  physiologic  saline  solution 
sterilized  by  filtration  and  so  diluted  that  the  initial  dose  will  contain 
0.0025  mg.  of  protein.  This  vaccine  maintained  its  therapeutic  proper- 
ties for  two  years. 

When  the  patient  can  be  studied  beforehand,  a  survey  of  his  habitual 
surroundings  should  be  made.  After  noting  all  the  flowering  plants 
which  might  reasonably  come  into  question,  skin  tests  should  be  made 
with  pollens  of  each  of  these  plants  in  order  to  determine  which  of  them 
are  responsible.  In  this  connection  it  must  be  remembered  that  pollens 
may  travel  great  distances  (Blackley) ,  and  a  field  of  grain  several  miles 
away  must  be  taken  into  account.  A  series  of  subcutaneous  injections 
with  the  proper  pollen  preparations  should  now  be  administered;  in 
my  experience  ten  or  more  should  be  given  with  gradually  increasing 
doses  at  intervals  of  five  or  seven  days,  and  the  course  of  treatment 
started  in  time  to  be  finished  a  week  or  two  before  the  expected  attack. 

If  the  attack  has  already  started,  treatment  should  be  begun  at  once 
with  a  vaccine  representing  the  pollens  most  likely  to  be  responsible 
for  the  attack.  Not  infrequently  relief  follows  within  twenty-four  hours 
after  an  injection.  The  injection  must  be  given  as  frequently  as  indi- 
cated to  relieve  the  symptoms  and  make  the  patient  fairly  comfortable. 

The  recent  reports  of  Clowes,3  Lovell,4  Lowdermilk,5  Ulrich,6  Koess- 

1  Jour.  Immunology,  1916,  1,  203. . 

2  Jour.  Lab.  and  Clin.  Med.,  1916,  1,  457.     (Excellent  review  of  the  literature.) 

3  Proc.  Soc.  Exper.  BioL  and  Med.,  1912-13,  x,  69. 

4  Lancet,  1912,  ii,  1716;  Practitioner,  1914,  xcii,  266. 

5  Jour.  Amer.  Med.  Assoc.,  1914,  Ixiii,  141. 

6  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1220. 


710  ACTIVE    IMMUNIZATION 

ler,1  Manning,2  Cooke,3  Wood,4  Goodale,5  and  Kitchens  and  Brown6 
indicate  that  the  vaccine  treatment  of  hay-fever,  and  particularly  the 
autumnal  variety,  is  frequently  successful  and  worthy  of  trial  not  only 
prophylactically  but  also  during  an  attack.  My  own  experience  has 
been  limited  to  7  cases  of  the  autumnal  type  due  to  rag-weed  sensiti- 
zation ;  4  of  these  persons  were  greatly  benefited,  1  was  not  aided  at  all, 
and  2  have  been  apparently  cured  for  a  period  of  about  two  years. 

Bronchitis. — Allen  speaks  very  highly  of  the  value  of  autogenous 
vaccines  in  the  treatment  of  acute  and  chronic  bronchitis  and  broncho- 
pneumonia  of  children.  Various  microorganisms  are  found  in  the  se- 
cretions, and  the  vaccines  used  are  generally  mixed.  The  usual  thera- 
peutic measures  are  employed  simultaneously.  It  is  claimed  that  vac- 
cines lessen  the  discomfort  and  hasten  recovery.  Recently  I  have  ob- 
served very  good  results  in  the  treatment  of  a  number  of  cases  of  chronic 
bronchitis  uncomplicated  by  bronchiectasis,  and  believe  that  an  au- 
togenous vaccine  prepared  under  proper  conditions  forms  a  valuable 
adjuvant  to  treatment. 

Pertussis. — A  hemophilic  bacillus  closely  resembling  the  influenza 
bacillus  has  been  ascribed  by  Bordet  and  Wollstein  as  the  cause  of  pertus- 
sis. Several  investigators  who  have  used  a  stock  vaccine  of  the  per- 
tussis bacillus  claim  that,  used  in  small  and  appropriate  doses,  the 
severity  of  the  paroxysms  of  coughing  is  lessened,  and  the  whole  course 
of  the  infection  shortened;  besides  this,  they  assert,  it  decreases  the 
danger  of  a  complicating  bronchopneumonia.  When  the  disease  is 
unusually  severe  and  the  prognosis  is  bad,  a  vaccine  may  be  administered 
in  doses  of  25,000,000  if  the  patient  is  over  four  years  of  age.  The 
pneumococcus  and  Bacillus  influenzse  are  frequently  associated,  and  a 
mixed  vaccine  of  these  microorganisms  may  be  of  special  aid  in  the  later 
stages  of  the  disease.  The  usual  remedial  measures  should  be  employed 
while  vaccines  are  being  tried.  The  reports  of  Bamberger7  and  Graham8 
indicate  that  the  administration  of  a  vaccine  may  be  of  value  in  lessening 
the  severity  of  the  disease.  I  have  treated  6  cases  with  indefinite 
results;  in  2  an  .autogenous  vaccine  was  employed  and  a  stock  vaccine  in 
the  remainder.  A  stock  vaccine  has  been  advocated  for  purposes  of 
prophylactic  immunization,  especially  in  institutions,  where  pertussis 
among  children  claims  a  high  mortality. 

1  Illinois  Med.  Jour.,  1914,  xxiv,  120.          2  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  655. 
3  Laryngoscope,  1915,  xxv,  108.  4  Chicago  Med.  Rec.,  1915,  xxxvii,  453  . 

5  Boston  Med.  and  Surg.  Jour.,  1915,  clxxiii,  42. 

6  Jour.  Lab.  and  Clin.  Med.,  1916,  1,  457.     (Excellent  review  of  the  literature.) 

7  Amer.  Jour.  Dis.  Children,  1913,  v,  33.        8  Amer.  Jour.  Dis.  Children,  1912,  iii,  41. 


ACTIVE    IMMUNIZATION    FOR   THERAPEUTIC    PURPOSES  711 

Otitis  Media. — Autogenous  vaccines  prepared  from  carefully  made 
cultures  of  the  diseased  tissues,  secured  with  the  aid  of  an  ear  speculum, 
may  prove  of  some  value  in  the  treatment  of  subacute  and  chronic 
suppurative  otitis  media.  Coates  has  reported  very  good  results. 
Additional  treatment  may  be  carefully  given,  but  not  infrequently 
more  harm  than  good  is  done  by  careless  flushing  and  cleansing  of  the 
auditory  canal,  whereby  deeper  and  healthy  tissues  become  infected. 
Free  drainage  should  be  afforded,  and  in  chronic  otitis  media  necrosed 
ossicles  and  granulations  may  require  surgical  removal.  Injections 
may  be  given  every  five  to  seven  days.  A  slight  increase  in  discharge 
after  the  first  one  or  two  doses  is  of  good  import,  and  indicates  a  slight 
focal  reaction.  With  Weston,  I  have  treated  a  large  number  of  cases  of 
suppurative  otitis  media  following  scarlet  fever,  and  whale  the  results 
were  seldom  brilliant,  in  general,  the  duration  and  severity  of  the  infec- 
tions were  favorably  influenced. 

TYPHOID  FEVER 

While  killed  preparations  of  the  typhoid  bacillus  have  been  injected 
soibcutaneously  as  a  method  of  treatment  hi  typhoid  fever  since  the 
work  of  Fraenkel1  in  1913,  the  subject  is  still  in  the  experimental  stage. 
An  analysis  of  the  literature  upon  this  subject  by  Callison,2  Watters,3 
and  Krumbhaar  and  Richardson4  show  that  at  best  the  ordinary  type 
of  heat-killed  vaccine  administered  subcutaneously  does  no  harm  and 
may  shorten  the  course  of  the  disease,  prevent  fever  relapses  and  compli- 
cations and  yield  a  slightly  lower  mortality.  The  treatment  should  be 
given  early  if  at  all,  while  the  resistance  and  general  condition  of  the 
patient  is  good.  The  injections  are  given  subcutaneously  and  the  doses 
should  be  modified  in  individual  cases  in  order  to  avoid  severe  reactions. 
The  initial  dose  may  be  100,000,000  bacilli;  three  days  later  250,000,000  or 
500,000,000  may  be  injected,  followed  by  three  or  four  similar  doses  at 
intervals  of  five  days.  During  convalescence  two  or  more  additional 
doses  may  be  given  in  an  effort  to  -prevent  relapses.  Garbat5  has  found 
the  subcutaneous  administration  of  sensitized  typhoid  bacilli  or  sero- 
bacterin  superior  to  the  non-sensitized  vaccine. 

More  striking  results  have  been  reported  by  Ichikawa,6  Kraus,7  and 
others  from  intravenous  injections  of  sensitized  vaccine.  Gay  and 

1  Deutsch.  Med.  Wchnschr.,  1893,  xix,  985. 

2  Amer.  Jour.  Med.  Sci.,  1912,  cxliv,  350.  3  Med.  Rec.,  1913,  Ixxxiv,  518. 

4  Amer.  Jour.  Med.  Sci.,  1915,  cxlix,  406. 

5  Jour.  Amer.  Med.  Assoc.,  1915,  64,  489. 

6  Ztsch.  f .  Immunitatsf.,  orig.,  1914,  xxiii,  32. 

7  Wien.  klin.  Wchnschr.,  1914,  xxvii,  1443. 


712  ACTIVE    IMMUNIZATION 

Chickering1  have  reported  upon  the  treatment  of  53  cases  of  typhoid 
fever  with  intravenous  injections  of  Vo"  to  2T  milligram  of  a  sensitized, 
polyvalent,  killed  typhoid  vaccine  sediment  prepared  after  the  method 
of  Gay  and  Claypoole.  The  mortality  was  9  per  cent.;  in  66  per  cent, 
of  the  cases  a  distinct  benefit  was  obtained,  as  shown  by  lowered  temper- 
ature, disappearance  or  amelioration  of  subjective  symptoms,  and  an 
apparently  accelerated  recovery.  These  effects  have  been  attributed 
to  the  production  of  hyperleukocytosis  and  various  antibodies  in  the 
patient's  blood. 

In  the  treatment  of  complications,  as  periostitis,  glandular  suppura- 
tion/ cholecystitis,  and  similar  complications  due  to  localized  infections, 
a  typhodd  vaccine  may  be  of  considerable  aid.  The  vaccine  should  be 
autogenous  if  possible,  and  mixed  if  the  lesions  are  open  and  other  bac- 
teria of  probable  pathogenic  activity  are  found.  Stone2  has  reported 
favorably  upon  the  treatment  of  cases  of  typhoid  carriers  with  the 
ordinary  vaccine,  and  it  would  appear  that  this  form  of  treatment 

is  worthy  of  trial. 

PNEUMONIA' 

While  pneumococcus  vaccine  has  been  employed  by  a  large  number 
of  physicians  in  the  treatment  of  lobar  pneumonia,  the  curative  value 
of  this  procedure  is  very  doubtful.  Stoner,8  Allen,4  Morgan,5  Harris,6 
and  others  have  reported  favorably  upon  the  vaccine  treatment  of  pneu- 
monia as  based  upon  mortality  statistics  and  clinical  impressions; 
Charteris,7  on  the  other  hand,  failed  to  observe  beneficial  results.  Rose- 
now  and  Hektoen8  have  prepared  a  vaccine  of  partialty  autoloyzed 
pneumococci  which  they  believe  proved  of  value  in  the  treatment  of 
pneumonia.  In  view  of  the  fact  that  different  types  or  strains  of  pneu- 
mococci may  produce  the  disease,  a  vaccine  if  employed  at  all  should  be 
autogenous;  if  a  stock  vaccine  is  employed,  it  should  be  polyvalent 
and  contain  at  least  type  strains  I  and  II.  It  is  difficult  to  find  support 
for  active  immunization  in  such  an  acute  and  relatively  brief  disease  as 
lobar  pneumonia,  and  if  a  vaccine  is  employed  it  should  be  given  as 
early  as  possible.  In  the  treatment  of  delayed  resolution  and  empyema, 
autogenous  vaccines  prepared  of  cultures  secured  from  the  sputum  or 
by  puncture  may  be  of  considerable  value. 

1  Archiv.  Int.  Med.,  1916,  xvii,  303.     (This  paper  contains  a  very  good  review  of 
the  literature  upon  the  treatment  of  typhoid  fever  with  the  intravenous  administra- 
tion of  sensitized  vaccines.) 

2  Jour.  Amer.  Med.  Assoc.,  1910,  Iv,  1708;  Amer.  Jour.  Med.  Sci.,  1912,  cxliii,  544. 

3  Amer.  Jour.  Med.  Sci.,  1911,  cxli,  186. 

4  Vaccine  Therapy  and  Opsonic  Treatment,  Blakiston,  Philadelphia,  1913. 

6  Proc.  Roy.  Soc.  Med.,  1910,  iii,  Suppl.  65.       6  Brit.  Med.  Jour.,  1909,  1,  1530. 

7  Glasgow  Med.  Jour.,  1912,  Ixxvii,  19.     8  Jour.  Amer.  Med.  Assoc.,  1913,  Ixi,  2203. 


TUBERCULOSIS — TUBERCULIN  THERAPY*  713 

OTHER  ACUTE  GENERAL  INFECTIONS 

Autogenous  vaccines  may  also  be  of  service  in  the  treatment  of 
puerperal  sepsis  and  ulcerative  endocarditis,  especially  after  the  more 
acute  symptoms  have  subsided.  In  the  former  condition  a  strepto- 
coccus may  be  obtained  by  blood  culture  or  by  intra-uterine  cultures; 
in  the  latter,  the  infecting  microorganism  is  obtained  only  by  blood 
culture.  Stock  vaccines  may  be  administered,  but  are  not  likely  to 
prove  of  value,  as  both  infections  are  usually  caused  by  streptococci, 
pneumococci,  or  some  similar  microorganisms  showing  so  much  differ- 
ence in  individual  properties  as  to  make  the  use  of  autogenous  vaccines 
imperative.  The  initial  doses  should  be  small — not  over  50,000,000 
cocci;  they  may  be  repeated  every  three  to  five  days,  and  are  gradu- 
ally increased  as  the  conditions  warrant. 

In  bacillary  dysentery  vaccine  treatment  has  not  given  particularly 
favorable  results.  If  employed  in  treatment  the  vaccine  should  be  pre- 
pared from  the  microorganism  isolated  from  the  feces  of  the  patient. 


TUBERCULOSIS. -TUBERCULIN  THERAPY 

Historic. — Within  the  last  twenty  years  the  subject  of  tuberculin 
therapy  has  elicited  considerable  discussion  in  the  diagnosis  and  treat- 
ment of  tuberculosis.  The  wide-spread  prevalence  of  the  disease,  not 
only  in  man,  but  in  the  lower  animals  as  well,  the  distressing  symptoms, 
the  gloomy  prognosis,  and  the  economic  importance  it  possesses,  are  a 
few  of  the  factors  that  have  stimulated  investigators  the  world  over  to 
zealous  and  persistent  efforts  directed  toward  discovering  a  means  of 
preventing  and  curing  this  great  scourge.  Owing  to  the  nature  of  the 
infection,  which  covers  relatively  long  periods  of  time,  and  the  fact  that 
much  time  is  required  for  the  conduct  of  experimental  studies,  researches 
are  of  necessity  prolonged,  tedious,  and  difficult.  Within  a  period  of 
less  than  six  years  the  cause  of  syphilis  has  been  discovered  and  isolated, 
and  valuable  diagnostic  reactions  and  a  well-nigh  specific  therapy  have 
been  devised.  The  discovery  of  an  early  and  specific  diagnostic  and 
therapeutic  measure  for  tuberculosis  will  achieve  still  greater  triumphs — 
in  fact,  few  could  be  greater  or  more  beneficial. 

Koch  was  the  first  to  note  the  curative  action  of  tuberculin,  and  it 
may  be  well  to  refer  here  to  the  original  description  of  his  fundamental 
experiments,1  which  have  been  the  basis  as  well  as  the  starting-point 
of  the  entire  study  of  tuberculins. 

1  Deutsch.  med.  Wochenschr.,  1891,  xvii,  101. 


714  ACTIVE    IMMUNIZATION 

"When  one  vaccinates  a  healthy  guinea-pig  with  a  pure  culture  of 
tubercle  bacilli,  the  wound,  as  a  rule,  closes  and  in  the  first  few  days 
seems  to  heal.  However,  in  from  ten  to  fourteen  days  a  hard  nodule 
appears  which  soon  breaks  down,  leaving  an  ulcer  that  persists  to  the 
time  of  death  of  the  animal.  There  is  quite  a  different  sequence  of 
events  when  a  tuberculous  guinea-pig  is  vaccinated.  For  this  experi- 
ment animals  are  best  suited  that  have  been  successfully  infected  four 
to  six  weeks  previously.  In  such  an  animal  the  inoculation  wound 
likewise  promptly  unites.  However,  no  nodule  forms,  but  on  the  next 
or  second  day  after  a  peculiar  change  occurs.  The  point  of  inoculation 
and  the  tissues  about,  over  an  area  of  from  0.1  to  1  cm.  in  diameter, 
grow  hard  and  take  on  a  dark  discoloration.  Observation  on  subsequent 
days  makes  it  more  and  more  apparent  that  the  altered  skin  is  necrotic. 
It  is  finally  cast  off,  and  a  shallow  ulceration  remains,  which  usually 
heals  quickly  and  permanently  without  the  neighboring  lymph-glands 
becoming  infected.  Inoculated  tubercle  bacilli  act  very  differently 
then  upon  the  skin  of  healthy  and  tuberculous  guinea-pigs.  This 
striking  action  is  not  restricted  to  living  tubercle  bacilli,  but  is  equally 
manifested  by  dead  bacilli,  whether  they  be  killed  by  exposure  to  low 
temperature  for  a  long  time  or  to  the  boiling  temperature,  or  by  the 
action  of  various  chemicals. 

"After  having  discovered  these  remarkable  facts,  I  followed  them  up 
in  all  directions  and  was  further  able  to  show  that  killed  pure  cultures  of 
tubercle  bacilli  ground  up  and  suspended  in  water  can  be  injected  in 
large  amounts  under  the  skin  of  healthy  guinea-pigs  without  producing 
any  effect  other  than  local  suppuration.  Tuberculous  guinea-pigs,  on 
the  other  hand,  are  killed  in  from  six  to  forty-eight  hours,  according  to 
the  dose  given,  by  the  injection  of  small  quantities  of  such  a  suspension. 
A  dose  which  just  falls  short  of  the  amount  necessary  to  kill  the  animal 
may  produce  extensive  necrosis  of  the  skin  about  the  point  of  injection. 
If  the  suspension  be  diluted  until  it  is  just  visibly  cloudy,  the  injected 
animals  remain  alive,  and  if  the  administration  is  continued  with  one 
to  two  day  intervals,  a  rapid  improvement  in  their  condition  takes 
place;  the  ulcerating  inoculation  wound  becomes  smaller  and  is  finally 
replaced  by  a  scar,  a  process  that  never  occurs  without  such  treatment; 
the  swollen  lymph-glands  become  smaller;  the  nutrition  improves, 
and  the  disease  process,  unless  it  is  too  far  advanced  and  the  animals 
die  of  exhaustion,  comes  to  a  standstill. 

"Thus  was  established  the  basis  for  a  rational  treatment  of  tuber- 
culosis. However,  such  suspensions  of  killed  tubercle  bacilli  are  un- 


TUBERCULOSIS TUBERCULIN    THERAPY  715 

suitable  for  practical  use,  since  they  are  neither  absorbed  nor  disposed  of 
in  other  ways,  but  remain  a  long  time  unaltered  at  the  point  of  inocula- 
tion and  occasion  smaller  or  larger  abscess. " 

Koch  showed  further  that  while  the  injection  of  tuberculous  guinea- 
pigs  with  large  doses  of  tubercle  bacilli  produced  rapid  death,  frequently 
repeated  small  doses  exerted  a  favorable  effect  upon  the  site  of  infection 
and  the  general  condition  of  the  animals.  The  same  observer  also 
realized  that  the  harmful  effects  of  injections  of  dead  tubercle  bacilli 
were  due  to  the  non-absorbable  parts  of  the  bacilli.  He  attempted  to 
extract  the  immunizing  substances,  and  in  this  way  produced  his  first 
or  Old  Tuberculin.  When  injected  into  tuberculous  guinea-pigs,  old 
tuberculin  produced  a  rapid  general  reaction  without  any  local  necrosis 
or  sloughing,  whereas  when  injected  into  a  healthy  guinea-pig,  no  re- 
action, either  local  or  general,  was  produced.  The  fact  that  the  general 
results  produced  by  old  tuberculin  were  analogous  to  those  obtained  by 
his  first  vaccine,  except  that  local  necrosis  did  not  occur,  induced  Koch, 
in  1891,  to  promulgate  it  as  a  specific  cure  for  tuberculosis  in  human 
beings. 

It  is  hardly  necessary  to  describe  the  hopeful  anticipation  with  which 
it  was  received,  and  the  keen  disappointment  that  followed  its  earlier 
clinical  use.  .Indiscriminate  use,  extravagant  expectations,  and  ex- 
cessive dosage  combined  to  yield  results  so  discouraging  as  to  swing  the 
pendulum  of  medical  opinion  so  far  the  other  way  that  even  now  the 
very  word  "  tuberculin "  suggests  to  many  minds  failure,  and  something 
to  be  avoided. 

A  few  earlier  followers  of  Koch  continued  their  studies  in  the  endeavor 
to  discover  the  causes  of  failure  in  tuberculin  therapy.  Their  researches 
have  led  to  new  principles  in  treatment  and  to  more  exact  knowledge  of 
its  indications,  as  well  as  its  contraindications.  As  now  employed,  its 
use  being  restricted  to  suitable  patients  and  administered  in  safe 
graduated  doses,  and  accepting  as  evidence  only  the  statements  of  those 
who  have  used  tuberculin  and  not  of  those  who  believe  it  to  be  dangerous 
and  have  never  used  it,  one  deduction  is  justified:  that  while  tuberculin 
is  not  a  specific  "cure"  for  tuberculosis, — any  more  than  hygiene,  diet, 
and  climate  are  cures, — it  helps  to  arrest  the  disease  and  is  in  general 
a  useful  factor  in  the  treatment  of  certain  types  of  the  disease.  Clinical 
studies  have  shtfwn,  however,  that  immunization  of  the  tuberculous 
patient  is  frequently  a  difficult  procedure,  owing  to  the  fact  that  such 
patients  are  prone  to  develop  a  remarkable  state  of  hypersusceptibility, 


716  ACTIVE   IMMUNIZATION 

in  consequence  of  which  every  inoculation  will  produce  a  reaction  that 
may  be  injurious  to  the  patient. 

Preparation  of  Tuberculins. — The  knowledge  that  tubercle  bacilli 
and  their  secretions  as  seen  in  vitro  contain  both  desirable  and  undesir- 
able substances  has  led  Koch  and  others  to  adopt  different  methods  of 
preparing  tuberculin  in  the  endeavor  to  obtain  the  .desirable  immunizing 
principles  in  as  pure  a  state  as  possible. 

As  a  consequence,  a  large  number  of  preparations  have  been  advo- 
cated from  time  to  time,  all  of  which  are  said  to  possess  some  special 
properties  and  virtues.  All  tuberculins,  whatever  their  mode  of  pre- 
paration and  manufacture,  are  derived  from  cultures  of  the  tubercle 
bacillus.  So  numerous  have  the  tuberculins  become,  and  so  superior 
are  the  advantages  claimed  for  each  new  product  over  the  older  ones, 
both  for  diagnostic  and  for  therapeutic  purposes,  that  only  a  few  of 
those  possessing  special  interest  and  value  can  here  be  described. 

1.  Old  Tuberculin  (0.  T.). — This  is  Koch's  original  tuberculin,  and 
is  the  variety  regarded  by  many  as  the  most  useful  both  in  diagnosis 
and  in  treatment.  Its  manufacture  was  based  upon  the  principle,  that 
the  toxins  elaborated  by  the  bacilli  into  the  culture-medium  or  liberated 
by  disintegration  of  the  bodies  were  chiefly  concerned  in  stimulating 
body-cells  to  the  formation  of  antibodies.  Since  the  bacillary  bodies 
were  regarded  as  mainly  responsible  for  the  production  of  abscesses  at 
the  point  of  inoculation,  they  are  eliminated  by  a  process  of  filtra- 
tion. 

Old  tuberculin  is  prepared  as  follows:  Large  shallow  flasks  contain- 
ing 5  per  cent,  of  glycerin  alkaline  broth  are  inoculated  with  a  culture 
of  human  tubercle  bacilli  and  grown  at  body  temperature  for  from  six  to 
eight  weeks,  at  the  end  of  which  time  the  bacilli  have  grown  into  a  flat 
sheet  covering  the  surface  of  the  fluid  (Fig.  137).  The  entire  contents 
are  then  subjected  to  a  current  of  steam  over  a  water-bath  for  the  pur- 
poses of  sterilization  and  for  concentration  into  one-tenth  of  the  original 
volume.  The  glycerin,  which  is  not  evaporated,  thus  constitutes  50 
per  cent,  of  the  resulting  mixture.  The  bacilli  are  removed  by  filtration 
through  a  Berkefeld  or  Chamberland  filter.  The  filtrate  is  a  clear, 
brown  fluid,  of  a  characteristic  odor,  which  keeps  indefinitely  and  is 
ready  for  use. 

Koch  considered  the  soluble  toxins  of  the  bacillus  as  the  desirable 
immunizing  agents,  and  believed  that  the  endotoxins  were  responsible 
for  the  necrotic  effects.  Since,  however,  it  was  accepted  that  bacter- 
iolytic  substances  would  be  formed  only  after  the  injection  of  intact  or 


TUBERCULOSIS TUBERCULIN  THERAPY 


717 


fragmented  tubercle  bacilli, — with  their  contained  endotoxins, — Koch 
added  T.  R.  and  later  B.  E.  to  his  list,  in  order  to  make  the  production 
of  antibacterial  substances  still  more  complete.  Furthermore,  in  order  to 
obtain  as  varied  a  supply  of  antibodies  as  possible  the  use  of  several 
tuberculins,  such  as  old  tuberculin  and  bacillus  emulsion,  was  recom- 
mended for  use  in  the  same  patient. 

2.  New  Tuberculin  (Known  as  T.  R.  or  Tuberculin  Residue). — This 
was  the  next  tuberculin  to  be  promulgated  by  Koch,1  and  is  prepared  as 
follows:  Virulent  cultures  of  human  tubercle  bacilli  are  grown  in  flasks 
of  nutrient  glycerin  broth  for  from  four  to  six  weeks,  the  bacilli  being 
then  filtered  off  and  dried  in  a  vacuum.  One  gram  of  the  dried  tubercle 


FIG.  137. — PREPARATION  OF  TUBERCULIN. 

A  flask  of  bouillon  culture  of  tubercle  bacilli  (three  to  four  weeks).     Note  the  surface 

layer  of  bacilli  with  stalactite  formations. 

> 

bacilli  is  ground  in  an  agate  mortar  until  all  the  bacilli  have  been  broken 
up.  To  the  pulverized  mass  100  c.c.  of  distilled  water  are  added, 
and  the  mixture  is  then  centrifugalized.  The  clear  supernatant  fluid 
is  poured  off,  and  is  now  known  as  Tuberculin  Oberes  (T.  O.,  not  to  be 
confounded  with  O.  T.).  It  contains  substances  not  precipitable  by 
glycerin.  The  sediment  is  again  dried,  powdered,  taken  up  in  a  small 
amount  of  water,  centrifuged,  the  supernatant  fluid  poured  off,  and  the 
process  repeated  until  no  sediment  is  precipitated  except  that  composed 
of  gross  accidental  particles.  The  fluids  resulting  from  all  the  centrif- 
ugalizations,  except  the  very  first,  are  poured  together  and  the  total 
should  not  measure  more  than  100  c.c.  This  opalescent  fluid  is  preserved 
with  20  per  cent,  of  glycerin  and  is  known  as  T.  R.  It  should  contain 
1  Deutsch.  med.  Wochenschr.,  1897,  xxiii,  209. 


718  ACTIVE    IMMUNIZATION 

in  each  cubic  centimeter  2  milligrams  of  solids,  representing  10  milli- 
grams of  dried  tubercle  bacilli. 

3.  Bacillen  Emulsion  (B.  E.). — This  was  a  still  later  form  of  tuber- 
culin made  by  Koch,1  and,  as  its  name  indicates,  it  is  an  emulsion  of 
tubercle  bacilli.     The  culture  is  grown  as  for  0.  T.;    the  bacilli  are 
filtered  off,  ground,  but  not  washed,  and  one  part  of  the  pulverized 
material  emulsified  in  100  parts  of  distilled  water;    an  equal  part  of 
glycerin  is  then  added,  making  a  50  per  cent,  glycerin  emulsion,  each 
cubic  centimeter  of  which  contains  the  immunizing  substances  of  5  mg. 
of  dried  tubercle  bacilli.     Since  B.  E.  was  not  washed,  it  was  assumed 
that  it  would  retain  all  extractives  and  the  entire  contents  of  the  bodies 
of  the  tubercle  bacillus. 

While  Koch  was  preparing  these  various  tuberculins  others  were 
being  made,  one  of  which,  prepared  by  Denys,  is  used  extensively  at 
present  in  the  treatment  of  tuberculosis. 

4.  Bouillon  Filtrate  (B.  F.). — This  is  practically  Koch's  old  tuber- 
culin unheated.     It  is  prepared  in  the  same  manner  as  0.  T.,  except 
that  the-  bacillus-free  filtrate — a  clear  fluid  said  to  contain  only  the 
soluble  secretions  of  the  bacilli,  plus  the  metabolized  culture-medium — 
is  not  heated  or  concentrated  and  is  ready  for  use  without  any  further 
modification. 

5.  Beraneck's    Tuberculin. — This    is    a   preparation   for   which    its 
inventor  claims  only  minimal  toxicity  and  a  high  content  of  specific 
substances.     It  is  prepared  by  cultivating  the  bacilli  on  a  non-peptonized 
5  per  cent,  glycerin  bouillon  medium,  which  is  not  neutralized.     The 
filtrate  from  this  culture  is  known  as  T.  B.,  or  toxin  bouillon.     The 
residue  is  shaken  for  a  long  time  at  from  60°  to  70°  C.  with  1  per  cent, 
orthophosphoric  acid.     Equal  volumes  of  the  unheated  toxin  bouillon 
and  of  the  acid  extract  of  the  bacillary  bodies  are  combined  to  form  the 
whole  tuberculin. 

6.  Von  Ruck's  Watery  Extract* — This  tuberculin  has  been  widely 
used  in  the  United  States.     It  is  prepared  as  follows:    Concentrate  a 
culture  in  vacuo  at  55°  C.  to  one-tenth  volume  (this  requires  about  one 
month).     Filter  through   paper    and    then   through   porcelain.     Pre- 
cipitate with  an  acid  solution  of  sodic  bismuth  iodid.     Filter  and  neu- 
tralize  the    acid   solution.     Filter   again.     Precipitate   with    absolute 
alcohol  to  make  90  per  cent,  alcohol  and  filter.     Wash  the  precipi- 
tate with  absolute  alcohol.     Dry  the  precipitate   and  make  a  1  per 

1  Deutsch.  med.  Wochenschr.,  1901,  xxvii,  839. 

2  Zeitschr.  f.  Tuberk.,  1907,  xi,  493. 


TUBERCULOSIS — TUBERCULIN    THERAPY  719 

cent,  aqueous  solution.  Filter.  The  last  filtrate  is  von  Ruck's  tuber- 
culin. 

7.  Dixon's  Tubercle-bacilli  Extract.1 — Dixon  has  prepared  a  tuber- 
culin by  treating  cultures  of  tubercle  bacilli  with  ether  and  extracting 
in  salt  solution.  This  has  yielded  good  results  in  the  treatment  of  tuber- 
culosis. This  product  is  prepared  from  the  living  organisms.  The 
tubercle  bacilli  are  grown  on  5  per  cent,  glycerin  veal  broth  for  a  period 
of  from  six  to  eight  weeks  at  a  temperature  of  37.5°  C.  They  are  re- 
moved from  the  incubator  and  collected  on  hard  filter-paper.  The 
filtrate  of  glycerin  broth  on  which  they  are  grown  is  discarded.  An 
equal  quantity,  by  weight,  of  tubercle  bacilli  of  the  bovine  and  human 
type  is  used.  The  mass  of  organisms  is  partially  freed  from  excess  of 
moisture  by  placing  it  between  two  sterile  filter-papers,  after  which  it  is 
placed  in  a  dish  in  the  incubator  for  from  twenty-four  to  forty-eight 
hours.  The  dried  organisms  are  then  washed  in  an  excess  of  ether, 
which  is  allowed  to  act  until  it  has  removed  all  the  water  and  glycerin. 
The  organisms  are  then  subjected  to  an  excess  of  fresh  ether  and  washed 
in  this  for  six  hours,  to  soften  the  wax  of  the  bacilli.  This  fat  separates 
so  that  it  collects  at  the  bottom  of  the  vessel  and  is  removed  with  a 
Pasteur  pipet.  After  the  second  addition  of  ether  has  been  removed  the 
mass  is  allowed  to  dry  until  no  ether  odor  is  perceptible.  After  the  mass 
of  tubercle  bacilli  has  been  thoroughly  dried  it  is  ground  in  a  mortar  and 
suspended  in  physiologic  salt  solution  in  the  proportion  of  1  part  of  the 
ground  mass  to  5  parts  of  an  8  per  cent,  salt  solution.  This  suspension 
is  shaken  in  a  shaking  machine  for  from  eight  to  ten  hours,  and  is  then 
allowed  to  stand  for  several  days  at  room  temperature.  It  is  finally 
passed  through  impervious  bacteria  filters  several  times  and  the  filtrate 
examined  microscopically,  bacteriologically,  and  physiologically.  Cul- 
ture tests  are  made  to  determine  its  freedom  from  contaminating  organ- 
isms, and  guinea-pigs  are  inoculated  to  ascertain  that  it  contains  no 
living  tubercle  bacilli.  One  cubic  centimeter  of  this  extract  represents 
0.2  gm.  of  the  organisms,  and  is  known  as  the  stock  solution,  from  which 
serial  dilutions  are  made.  This  solution  is  sterile,  but  0.5  per  cent,  of 
phenol  (carbolic  acid)  is  added  as  a  preservative  to  prevent  subsequent 
contamination. 

While  the  tuberculins  just  described  are  those  mainly  used,  many 

others  have  been  prepared  and  advocated  in  the  diagnosis  and  treatment 

of  tuberculosis.     The  aim  is  always  to  obtain  the  specific  substances, 

with  as  little  as  possible  of  the  toxic  substances — not  only  those  con- 

1  Penna.  Health  Bull,  1914,  No.  28,  April. 


720  ACTIVE    IMMUNIZATION 

cerned  in  producing  necrosis,  but  likewise  the  protein  constituents 
responsible  for  specific  sensitizing  action  and  anaphylactic  disturbances. 
For  example,  tuberculocidin  and  tuberculol  are  examples  of  attempts  at 
isolating  the  pure  immunizing  principle;  endotin,  or  Moeller's  tuber- 
culin, is  an  example  of  an  endeavor  to  rid  the  culture  fluid  of  protein 
substances.1 

Action  of  Tuberculin. — If  a  healthy  or  a  tuberculous  individual  is 
injected  with  old  tuberculin,  an  immunity  will  be  established  only 
against  the  substances  contained  in  this  preparation.  That  this  does 
not  fulfil  the  requirements  is  proved  by  the  fact  that  an  animal  im- 
munized against  this  tuberculin  will  not  be  protected  against  a  later 
infection  with  living  tubercle  bacilli.  It  was  mainly  for  this  reason 
that  tubercle  emulsion  and  new  tuberculin  were  devised  and  used,  in 
the  effort  to  provide  immunization  not  only  against  the  products  of  the 
bacilli,  but  against  the  bacilli  themselves,  and  to  bring  about  their  actual 
destruction. 

Probably  none  of  the  various  tuberculins  can  be  considered  as  re- 
presenting the  true  toxins  of  the  tubercle  bacillus,  although  they  simu- 
late these  substances  with  sufficient  closeness  to  bring  about  partial 
immunity  against  some  of  the  poisonous  products  and  to  warrant  their 
use  in  tuberculosis.  An  individual  may  become  immunized  against  old 
tuberculin  so  that  large  doses  will  evoke  no  reaction,  but  this  does  not 
necessarily  imply  that  a  cure  has  resulted;  in  fact,  the  injection  of  another 
preparation,  such  as  new  tuberculin  or  bacillus  emulsion,  may  bring 
about  a  reaction. 

Partial  immunization  possesses,  however,  distinct  advantages,  in 
that  it  lessens  some  of  the  symptoms  of  tuberculosis.  In  addition, 
tuberculin  immunization  may  give  most  important  aid  in  walling  off 
tuberculous  foci  with  fibrous  tissue,  and  in  this  manner  bring  about  a 
condition  of  potential  cure.  Following  the  injection  of  tuberculin  a 
focal  reaction  occurs,  characterized  by  hyperemia  and  exudation  about 
the  diseased  tissues.  While  an  excessive  dose  of  tuberculin  may  produce 
excessive  hyperemia,  exudation,  and  necrosis  of  tuberculous  tissue  and 
lead  to  actual  harm,  these  being  some  of  the  effects  that  followed  the 
early  use  of  tuberculin  and  resulted  in  bringing  it  into  high  disfavor, 
smaller  and  carefully  graduated  doses  tend  to  produce  a  mild  inflamma- 
tory hyperemia  leading  to  destruction  of  tuberculous  tissue  and  the 

1  For  a  full  account  of  these  and  other  preparations  I  refer  the  reader  to  the  book 
of  Hamman  and  Wolman,  "  Tuberculin  in  Diagnosis  and  Treatment,"  1912,  Appleton 
&Co. 


TUBERCULOSIS — TUBERCULIN   THERAPY  721 

formation  of  connective  tissue  which  encapsulates  the  focus;  with  this 
there  is  also  associated  the  local  production  of  antibodies. 

The  Use  of  Living  Tubercle  Bacilli. — Any  vaccine  that  will  give 
complete  immunization  against  the  tubercle  bacillus  and  all  its  products, 
in  addition  to  a  healing  focal  reaction  of  the  right  degree,  will  prove 
of  the  greatest  value  in  active  prophylactic  and  curative  immunization. 

As  has  been  stated  repeatedly,  this  is  probably  best  obtained  by  using 
living  cultures  in  such  form  that  they  cannot  produce  the  disease. 
Obviously,  it  is  a  difficult  matter  to  find  such  a  culture  or  to  modify 
one  to  meet  the  requirements  at  hand.  When  this  problem  is  solved, 
it  may  then  be  possible  to  provide  universal  prophylactic  immunization 
and  to  aid  the  actually  diseased  in  overcoming  their  infection.  At 
present  the  tuberculins  are  not  adapted  for  prophylaxis  because  they 
possess  only  partial  immunizing  powers,  although  these  effects  may  be 
of  distinct  aid  to  the  tuberculous  patient,  in  addition  to  producing  a 
focal  reaction  of  value  in  walling  off  a  lesion  and  producing  local  anti- 
bodies. 

Probably  the  first  work  done  with  the  living  bacillus  was  that  of 
Dixon1  with  attenuated  cultures.  This  worker  found  that  animals 
inoculated  with  an  old  culture  containing  club-shaped  and  branching 
forms  of  tubercle  bacilli  would  resist  subsequent  inoculation  with  viru- 
lent organisms.  Since  then  numerous  investigators — Trudeau,2  Pear- 
son,3 de  Schweinitz,4  McFadyean,5  Levy,6  Pearson  and  Gilliland,7 
Behring,8  Thomassen,9  Neufeld,10  Theobald  Smith,11  Webb  and  Wil- 
liams,12 and  others — have,  either  directly  or  indirectly,  supported  this 
original  work,  thus  indicating  that  the  most  effectual  active  immuniza- 
tion is  secured  by  using  living  cultures.  The  method  employed  by  Webb 
and  Williams  is  worthy  of  special  mention,  inasmuch  as  the  ascending 
doses  of  bacilli  are  actually  counted  by  an  ingenious  method  devised  by 

1  Medical  News,  Philadelphia,  October  19,  1889. 

2  Amer.  Jour.  Med.  Sci.,  August,  1906;   June,  1907;   New  York  Med.  Jour., 
July  23,  1893;  Medical  News,  October  24,  1903. 

3  Proc.  First  Internat.  Vet.  Congress,  1893. 

4  Medical  News,  December  8,  1894. 

6  Jour,  of  Comparative  Path,  and  Therap.^June,  1901;    March,  1902. 

6  Centralbl.  f.  Bakt.,  1903. 

7  Phila.  Med.  Jour.,  November  29,  1902;   Univ.  of  Penna.  Med.  Bull.,  1905. 

8  Beitrage  z.  exper.  Therapie,  Reft.  s.  Marburg,  1902. 

9  Recuil  de  Med.  Vet.,  January  15,  1903. 

10  Deutsch.  med.  Wochenschr.,  September  1,  1902;   April  28,  1904. 

11  Jour.  Med.  Research,  June,  1908. 

12  Trans,  of  the  Sixth  Internat.  Congress  on  Tuberculosis,  1908;    Jour.  Med. 
Research,  1911,  xix,  1. 

46 


722  ACTIVE    IMMUNIZATION 

Barbour.1  These  observers  used  this  method  quite  extensively  with 
lower  animals,  and  have  also  secured  good  results  with  persons  willing 
and  anxious  to  take  all  possible  risks  for  the  possible  good  that  may 
result.  In  no  case  have  harmful  results  followed  the  injections. 

More  recently  the  profession  and  laity  have  been  agitated  by  the 
extravagant  claims  of  Friedmann  for  a  vaccine  of  living,  acid-fast 
bacilli  derived  from  the  turtle.  This  culture  is  said  to  be  avirulent  for 
human  beings,  and  to  be  capable  of  stimulating  specific  antibodies  and 
thus  bringing  about  a  cure.  The  unfortunate  methods  by  which  these 
claims  have  been  exploited,  and  the  more  recent  investigations,  tending 
to  show  that  no  good  has  followed  its  use,  and  that  the  vaccine  is  not 
entirely  harmless,  preclude  any  statements  at  this  time.  The  principle 
involved,  however,  namely,  the  use  of  a  living  vaccine  composed  of 
microorganisms  so  altered  by  passage  through  a  lower  animal  that  they 
cannot  produce  tuberculosis  in  the  human  being,  but  yet  resembling  the 
human  bacillus  closely  enough  to 'produce  specific  antibodies,  is  sound, 
and  should  stimulate  further  experimental  research  in  this  direction. 

Patients  Suitable  for  Tuberculin  Treatment. — In  deciding  which 
tuberculous  patients  are  best  suited  to  receive  tuberculin  treatment  it 
must  be  borne  in  mind  that  tuberculin  is  not  a  form  of  passive  immunity, 
depending  upon  antitoxins,  bacteriotropins,  and  bactericidins,  but  that 
it  serves  to  stimulate  the  body-cells  to  produce  these  protective  sub- 
stances. The  output  of  antibodies  provoked  by  tuberculin  is  dependent 
upon  the  condition  of  the  body  as  a  whole,  and  its  administration  can 
be  of  no  help  if  the  resources  of  the  body  are  exhausted  and  the  cells  are 
incapable  of  beneficiary  reaction.  In  other  words,  tuberculin  therapy 
must  be  guided  by  the  same  considerations  that  influence  the  vaccine 
treatment  of  acute  infections  in  general. 

1.  Patients  afflicted  with  incipient  tuberculosis  are  proper  subjects 
for  receiving  tuberculin  treatment,  since  it  tends  to  protect  them  from 
relapse,  and  insures,  to  a  greater  degree,  their  ability  to  continue  work. 

2.  Advanced  and  moderately  advanced  cases  may  be  given  tuberculin 
if  the  nutrition  is  fair,  the  febrile  reaction  mild,  the  pulse  not  very  rapid, 
and  if  the  treatment  is  controlled  by  rest.     Old  fibroid  cases  with  fair 
nutrition  are  especially  suitable,  as  such  patients  become  capable   of 
moderate  activity  and  are  much  less  likely  to  suffer  from  relapses. 

3.  Cases  of  tuberculosis  of  the  lymphatic  glands,  skin,  and  special 
organs  may  be  benefited  by  prolonged  and  careful  tuberculin  therapy. 

4.  Cases  of  latent  tuberculosis,  especially  the  children  of  infected 

1  The  Kansas  Univ.  Sci.  Bull.,  1907,  iv,  No.  1. 


TUBERCULOSIS TUBERCULIN    THERAPY  723 

families  who  are  below  par  physically  and  show  tuberculin  hypersen- 
sitiveness,  with  indefinite  physical  signs,  are  proper  subjects  for  re- 
ceiving tuberculin  treatment. 

5.  The  question  arises  as  to  whether  tuberculin  may  be  administered 
to  ambulant  patients.  Tuberculin  should  be  regarded  as  but  one  factor 
in  the  treatment  of  tuberculosis,  and,  as  such,  should  be  combined  with 
the  best  therapeutic  measures  available.  Therefore  the  tuberculin 
treatment  is  supplementary  to  rest,  hygiene,  and  fresh  air,  and  the 
benefit  of  the  sanatorium  should  not  be  denied  to  patients,  especially 
to  the  poorer  ones.  The  treatment  of  a  mildly  progressive  ambulant 
case  should  be  undertaken  only  when  prolonged  rest  in  bed  has  had  no 
visible  effect,  and  when  no  measures  can  be  devised  for  administering 
the  tuberculin  while  the  patient  is  in  bed,  as,  e.  g.,  patients  who  have 
been  at  the  sanatorium  and  have  returned  to  work,  or  those  who  cannot 
be  persuaded  to  enter  a  sanatorium  or  for  whom  no  place  can  be  found. 
The  tuberculin  treatment  of  more  chronic  or  localized  tuberculosis  may 
be  successfully  undertaken  in  the  clinic  or  office. 

Contraindications  to  Tuberculin  Therapy. — Owing  to  the  increased 
focal  hyperemia  that  follows  the  injection  of  tuberculin,  hemoptysis  has 
been  considered  a  contraindication  to  its  use.  In  such  cases  it  is  well 
to  wait  for  some  time  at  least  and  begin  the  injections  with  very  small 
doses,  as  the  ultimate  effect,  namely,  the  production  of  fibrous  tissue, 
may  be  of  great  aid  in  prolonging  life. 

Various  authorities  have  expressed  different  views  regarding  other 
contraindications,  such  as  marked  general  weakness,  fever,  cardiac 
disease,  nephritis,  epilepsy,  syphilis,  hysteria,  etc.  As  was  stated  by 
Hamman  and  Wolman,  these  are  not  contraindications,  but  unfortunate 
complications  that  would  embarrass  any  form  of  treatment.  Tuberculin 
may  be  given  to  any  patient  whose  resisting  powers  have  not  been  too 
much  depressed  as  the  result  of  complications.  For  the  beginner  in 
this  form  of  therapy,  however,  it  is  advisable  that  he  acquire  experience 
by  undertaking  the  treatment  of  uncomplicated  cases  before  assuming 
the  responsibility  of  treating  the  more  difficult  ones. 

The  fact  that  the  ophthalmic  test  has  been  made  is  no  contraindica- 
tion to  treatment  by  tuberculin  if  the  reaction  has  subsided,  since  a 
flare-up  rarely  occurs  except  after  large  diagnostic  doses  (Hamman  and 
Wolman). 

ADMINISTRATION  OF  TUBERCULIN 

1.  Subcutaneous  Injection. — Of  most  importance  in  this  connection 
is  the  attitude  of  the  therapeutist  toward  the  question  of  reactions 


724  ACTIVE    IMMUNIZATION 

following  the  administration  of  tuberculin.  He  must  know  whether 
he  does  or  does  not  wish  to  obtain  symptoms  of  a  tuberculin  reaction 
during  the  treatment;  the  size  of  the  initial  and  particularly  of  subse- 
quent doses  will  depend  upon  his  desire  to  obtain  a  reaction  or  upon  his 
anxiety  to  avoid  it. 

Reactions. — At  the  present  time  tuberculin  is  never  used  for  the 
purpose  of  obtaining  strong  reactions,  such  as  Koch  originally  insisted 
upon  getting.  Koch  administered  a  dose  large  enough  to  elicit  a  strong 
constitutional  reaction,  and  repeated  it  at  intervals  of  one  or  more  days 
until  that  dose  no  longer  produced  a  reaction,  after  which  a  still  larger 
dose  was  given  and  the  former  procedure  repeated.  Many — too  many — 
were  unable  to  pass  through  this  therapeutic  furnace  unsinged,  and,  in 
fact,  the  results  obtained  led  to  the  period  known  as  the  "  tuberculin 
delirium, "  ending,  as  Hamman  and  Wolman  stated,  "  to  the  consequent 
downfall  of  the  arrogant  therapy  to  an  humble  position,  whence  it  is 
but  just  emerging,  chastened  and  refined,  to  assert  its  modest  but  now 
truthful  claims  to  a  therapy  less  spectacular  but  more  healing,  less 
forceful  but  more  gently  persuasive:  healing  a  few,  helping  many,  and 
hur ting  none. " 

While  there  is  this  general  unanimity  of  opinion  regarding  the  harm- 
ful effects  of  strong  reactions,  yet  tuberculin  therapeutists  may  be 
divided  into  those  who  scrupulously  avoid  all  reaction,  those  who  are 
a  little  bolder  and  do  not  object  to  a  very  mild  reaction,  and  to  an 
intermediate  class.  In  the  first  group  are  Sahli  and  Trudeau;  the  former 
claims  that  it  is  essential,  first  of  all,  to  do  no  harm,  and  that  cases 
treated  cautiously  attain  a  tolerance  for  high  doses  as  soon  as,  and  even 
sooner  than,  those  that  are  rushed.  Petruschky  is  an  exponent  of  the 
bolder  method,  believing  that,  by  proceeding  very  cautiously,  much 
time  is  wasted  and  not  enough  focal  reaction  is  produced  to  promote 
healing.  To  the  intermediate  class,  and  approaching  rather  the  timid 
class,  are  Bandelier  and  Roepke,  Hamman  and  Wolman,  and  many 
others.  These  observers  adopt  no  scheme  of  fixed  dosage,  but  study  the 
individual  patient,  remembering  what  is  to  be  avoided,  rather  than  the 
high  dose  to  be  reached. 

The  constitutional  symptoms  of  a  reaction  are:  temperature,  loss  of 
weight,  rapid  pulse,  and  general  symptoms,  such  as  malaise,  headache, 
chilliness,  arthritic  pains,  gastric  or  intestinal  disturbances,  nausea,  loss 
of  appetite,  insomnia,  and  skin  eruptions. 

Of  these  general  signs,  the  most  important  are  fever,  loss  of  weight, 
and  symptoms  of  general  depression.  The  temperature  should  be  taken 


TUBERCULOSIS — TUBERCULIN   THERAPY  725 

for  several  days  preceding  the  initial  dose,  so  that  the  patient's  "  normal" 
limits  are  known  before  the  tuberculin  is  given.  When  the  usual  maxi- 
mum temperature  is  reached,  it  is  especially  necessary  to  watch  closely 
for  additional  signs  of  reaction.  Without  some  constitutional  dis- 
turbance a  slight  pyrexia  is  of  less  significance,  and  it  is  to  be  remembered 
that  tuberculous  patients  may  have  a  flare-up  of  fever  when  they  are 
not  being  treated  with  tuberculin.  Denys  refuses  to  consider  any 
temperature  reaction  as  due  to  tuberculin  that  comes  on  more  than 
forty-eight  hours  after  the  injection  has  been  given.  It  is  characteristic 
of  tuberculin  reactions  that  the  rise  is  abrupt,  and  not  in  step-like  pro- 
gression. Hamman  and  Wolman  would  hesitate  to  ascribe  an  elevation 
coming  on  suddenly  in  the  midst  of  a  perfectly  smooth  course  of  tuber-, 
culin  treatment,  and  unaccompanied  by  a  local  reaction,  to  the  injections, 
when  the  dose  has  not  been  unduly  large. 

Loss  of  weight  is  a  delicate  sign — more  valuable  as  a  symptom  of 
overdosage  late  in  the  treatment  than  as  a  protection  against  the  sud- 
denly appearing  reactions. 

Bandelier  and  Roepke  regard  an  increase  in  the  pulse-rate  as  a  sign 
of  great  importance.  Hamman  and  Wolman  and  Lawrason  Brown 
have  not  been  able  to  observe  this  sign  very  frequently. 

The  local  signs  are:  Pain,  tenderness,  and  swelling  at  the  site  of 
injection.  These  may  consist  of  all  gradations  from  simple  thickening  of 
the  skin  to  a  wide,  deep,  hard,  and  painful  node,  with  or  without  in- 
volvement of  the  neighboring  lymphatics. 

The  local  reaction  has  assumed  great  importance  in  recent  years, 
especially  since  Denys  drew  attention  to  it  as  serving  as  a  warning  of  the 
approach  of  a  general  reaction.  It  is  characterized  by  the  development 
of  pain,  tenderness,  and  redness  about  the  site  of  a  former  injection. 
It  is  more  often  solitary  than  any  other  sign,  and,  in  the  absence  of  a 
temperature  record,  it  is  safe  to  proceed,  the  sole  guidance  being  the 
local  reaction,  both  subjective  and  objective.  A  large  dose  of  tuber- 
culin  may  give  a  local  reaction,  due  merely  to  its  bulk  and  concentra- 
tion (500  to  600  mg.),  simulating  a  true  reaction;  this  may  be  avoided 
by  dividing  the  dose  into  two  injections,  which  are  given  at  the  same 
time,  but  not  into  neighboring  areas  of  skin. 

The  focal  signs  in  pulmonary  tuberculosis  are:  increased  cough, 
dyspnea,  expectoration,  thoracic  pain,  hemoptysis,  and  extension  of 
the  physical  signs. 

Focal  signs  of  a  reaction  are  an  indication  that  the  treatment  has 
been  conducted  too  rapidly. 


726  ACTIVE   IMMUNIZATION 

Unless  one  belongs  to  the  ultra-conservative  class  of  tuberculin 
therapeutists,  it  is  not  a  slight  focal  reaction  that  is  to  be  avoided,  but 
those  reactions  that  are  large  enough  to  manifest  themselves  by  changes 
in  the  physical  signs  or  by  decided  symptoms.  On  the  contrary,  it  is  the 
production  of  such  slight  hyperemia  about  the  focus  of  disease  that 
constitutes  the  most  valuable  result  of  tuberculin  therapy.  It  is  well  to 
start  with  a  minute  dose,  and  push  the  dosage  rapidly  until  a  slight  focal 
reaction  at  the  site  of  injection  or  mild  pyrexia  is  observed.  When  a 
mild  focal  reaction  is  not  produced,  the  patient  is  not  receiving  his  due 
amount. 

Dosage. — Since  each  patient  is  a  law  unto  himself,  the  initial  dose 
should  be  so  small  that  no  harm  can  result  from  its  use.  White  and  Van 
Norman  make  the  initial  dose  equal  to  the  quantity  of  tuberculin 
which,  when  applied  cutaneously,  will  elicit  a  minimal  reaction  after 
seventy-two  hours. 

As  regards  the  size  of  the  initial  dose,  patients  can  be  divided  into 
three  classes:  (1)  Children;  (2)  patients  who  exhibit  a  slight  pyrexia 
or  are  not  in  good  condition;  (3)  patients  in  good  condition.  The 
following  table  of  doses  is  that  given  by  Hamman  and  Wolman,  the 
smaller  initial  dose  being  for  classes  1  and  2,  the  larger  for  class  3. 

TABLE  26.— INITIAL  AND  MAXIMAL  DOSES  OF  THE  COMMONLY  USED 

TUBERCULINS 


TUBERCULIN 

INITIAL  DOSE 

MAXIMAL  DOSE 

O.  T.. 
T.  R  
B.  E..  . 

0.0000001    c.c.  to  0.000001    c.c. 
0.000001      c.c.  to  0.0001        c.c. 
0  000001      c  c  to  0  0001        c  c 

1  C.C. 

2  c.c. 

2P   C 

B.  F.  .. 

0.00000001  c  c  to  0  0000001  c  c 

Beraneck's  

Of  A/32,  0.05  c.c. 

Of  H,  1  c.c. 

Old  tuberculin  (0.  T.)  is  put  up  in  ampules  holding  1  c.c.  and  5  c.c. 
From  these,  higher  dilutions  are  prepared  by  adding  sterile  normal  salt 
solution,  using  sterile  glassware,  starting  with  a  1 :  10  (A)  of  the  original 
strength,  then  making  a  1 :  10  from  this  (B),  a  1 :  10  from  that  (C),  a 
1 :  10  from  that  (D),  and  so  on  to  the  desired  dilution.  As  1  c.c.  of  the 
original  product  represents  1000  milligrams  of  the  pure  tuberculin,  1  c.c. 
of  dilution  A  will  contain  100  milligrams;  B,  10  milligrams;  C,  1  milli- 
gram; D,  0.1  milligram;  E,  0.01  milligram,  and  F,  0.001  milligram, 
which  is  the  higher  of  the  initial  doses  just  given. 

New  tuberculin  (T.  R.)  is  so  prepared  that  1  c.c.  represents  5  milli- 
grams of  the  dry  powder.  Dilutions  may  be  prepared  in  a  manner 


TUBERCULOSIS — TUBERCULIN   THERAPY  727 

similar  to  the  foregoing  by  diluting  the  original  product  1 :  10  (A)  ; 
1  c.c.  of  this  is  in  turn  diluted  to  1 :  10  (B);  of  this,  1 :  10  (C);  and  of 
this,  1 :  10  (D);  1  c.c.  of  A  contains  0.5  milligram;  1  c.c.  of  B,  0.05; 
1  c.c.  of  C,  0.005,  and  1  c.c.  of  D,  0.0005  milligram. 

Beraneck's  tuberculin  is  marketed,  ready  diluted,  in  a  series  of  syringes, 
A/128,  A/64,  A/32,  to  A/4,  A/2,  A,  B,  C,  to  H.  H  is  the  pure  tuber- 
culin. Each  solution  is  one-half  the  strength  of  the  next  stronger  one. 
The  increase  of  dosage  is  usually  by  0.1  c.c.  until  0.5  c.c.  is  given.  Then 
0.1  c.c.  of  the  next  stronger  dilution  is  given,  and  so  on. 

Dixon's  tuberculin  is  supplied  in  syringes  in  the  series  of  dilutions 
given  in  the  following  table,  so  that  the  doses  may  be  increased  as  is 
found  desirable : 

TABLE  27.— ADMINISTRATION   OF  DIXON'S  TUBERCULIN 

AMOUNT  (IN  GRAMS)  OF  AMOUNT  (IN  GRAMS)  OF 

DILUTION  EXTRACT  OF  TUBERCLE  DILUTION  EXTRACT  OF  TUBERCLE 

No.  BACILLI  CONTAINED  IN  No.  BACILLI  CONTAINED  IN 

1 0.001  11 0.1  ' 

2 0.01  12 0.11 

3 0.02  13 0.12 

4 0.03  14 0.13 

5 0.04  15 0.14 

6 0.05  16 0.15 

7 .0.06  17 0.16 

8 0.07  18 0.17 

9 0.08  19 0.18 

10 0.09  20 0.19 

It  is  suggested  that  in  ordinary  cases  the  injection  of  each  dilution  be 
repeated  at  least  five  times  before  changing  to  the  next  number  or  next 
stronger  dilution. 

As  a  general  rule,  the  succeeding  doses  of  a  tuberculin  may  be  in- 
creased by  0.1  c.c.,  due  care  being  exercised  when  the  dilutions  are 
changed.  If  the  patient  is  quite  sensitive,  it  may  be  well  to  repeat  the 
first  dose  of  each  stronger  solution  once  or  more  often  as  it  is  reached, 
and,  instead  of  proceeding  to  0.2  c.c.,  give  only  0.15  c.c.,  thus  tiding  over 
the  gap.  If  the  patient  is  not  so  sensitive,  a  few  leaps  may  be  taken 
with  the  weaker  dilution,  so  as  to  test  the  patient's  tolerance,  and,  if  it 
is  good,  the  next  higher  dilution  is  given  at  once. 

Site  of  Injection. — Injections  are  probably  best  given  in  the  back, 
at  the  lower  angle  of  the  scapula.  Local  reactions  are  more  reliable 
here  than  when  the  injections  are  given  in  the  arm.  All  injections 
should  be  given  under  aseptic  precautions  and  with  a  sterilized  syr- 
inge, in  exactly  the  same  manner  as  any  bacterial  vaccine  is  given. 
All  injections  should  be  subcutaneous;  intramuscular  injections  are 


728  ACTIVE   IMMUNIZATION 

to  be  avoided,  and  no  great  advantage  is  to  be  gained  from  using  intra- 
venous injections. 

Time  of  Injection. — There  is  some  difference  of  opinion  as  to  the  best 
time  of  the  day  for  administering  tuberculin  therapeutically.  If  given 
in  the  morning,  a  slight  febrile  reaction  may  occur  during  the  evening 
which  would  otherwise  be  overlooked.  Brown  is  in  favor  of  the  after- 
noon as  the  most  suitable  time,  because  it  affords  an  opportunity  for 
omitting  the  dose  in  case  there  is  an  accidental  rise  of  temperature  on 
that  day.  On  the  other  hand,  it  is  contended  that  the  rest  at  night 
would  tend  to  prevent  the  occurrence  of  the  reactions  that  might  appear 
if  the  patient  were  up  and  about. 

While  it  is  not  essential  that  the  patient  rest  for  a  few  hours  after  a 
dose  has  been  administered,  this  is  advisable,  and  where  absolute  rest 
can  be  enforced,  the  dosage  may  be  increased  with  greater  rapidity  than 
in  ambulant  patients. 

Interval  Between  Doses. — The  interval  between  injections  is  usually 
from  three  to  four  days — i.  e.^fwo  Injections  a  week.  This  interval  is 
merely  tentative,  and  while  it  should  not  be  shortened,  it  may  be  nec- 
essary to  prolong  it.  While  most  reactions  set  in  within  from  twenty- 
four  to  thirty-six  hours,  some  may  begin  as  late  as  from  forty-eight  to 
sixty  hours  (L.  Brown).  By  waiting  at  least  three  days  we  may  be 
assured  that,  if  no  reaction  has  occurred,  none  will  take  place. 

The  usual  interval  is  maintained  as  long  as  the  patient  is  doing  well. 
After  a  while  the  patient  becomes  intolerant  and  exhibits  slight  reac- 
tions, depression,  and  loss  of  weight.  In  such  instances  the  interval  may 
be  increased  to  a  week  and  the  injections  continued.  Hamman  and  Wol- 
man  find  this  occurrence  so  frequent  that  they  advise  creasing  the 
dose  interval  to  one  week,  when  the  dose  of  100  mg.  of  0.  T.  is  reached, 
200  mg.  of  T.  R.  and  B.  E.,  and  50  mg.  of  B.  F. 

2.  Other  Routes  for  the  Administration  of  Tuberculin. — Oral 
Route. — It  has  been  shown  that  reactions  may  follow  the  oral 
administration  of  tuberculin.  But  absorption  is  so  irregular  that  a 
quantity  of  tuberculin  may  be  absorbed  suddenly  and  cause  unexpected 
reactions.  Much  depends,  apparently,  upon  the  state  of  digestion 
and  upon  the  condition  of  the  alimentary  tract.  The  oral  route  also 
deprives  the  physician  of  the  benefits  to  be  obtained  from  using  the  local 
reaction  as  a  guide.  Otherwise  the  method  is  simple  and  the  tuber- 
culin may  be  administered  in  the  form  of  tablets  or  in  capsules.  It 
is  important,  however,  to  exercise  supervision  over  the  patient.  S. 


TUBERCULOSIS — TUBERCULIN   THERAPY  729 

Solis  Cohen  has  used  a  modification  of  Latham's  method,  and  reports 
favorable  results:  "Tuberculin  residue  (T.  R.)  triturated  with  milk 
sugar  is  given  with  skim  milk,  whey,  or  beef-juice.  The  initial  dose  is 
0.000001  mg.  Both  subjective  and  objective  symptoms  of  reaction  are 
watched  for.  The  dose  is  repeated  once  or  twice  weekly,  according  to 
results.  It  is  gradually  increased  by  increments  of  0.000001  mg.  to  the 
reaction  point,  and  then  dropped  one  point  lower,  and  so  continued  for 
some  weeks.  Later,  a  further  increase  is  attempted,  and  if  reaction  is 
not  shown,  is  proceeded  with  in  a  similar  gradual  way.  The  arbitrary 
increment  of  0.000001  mg.  is  maintained  during  this  remittent  progres- 
sion until  0.0001  mg.  has  been  reached.  After  that  the  increment  may 
be  raised  to  0.00001  mg.  Thus,  by  successive  stages,  a  maximum  dose 
is  attained  at  a  point  determined  for  each  individual  by  all  the  factors 
in  the  case,  including  the  rapidity  of  increase,  character  and  intensity  of 
reaction,  and  maintenance  of  tolerance,  as  well  as  the  focal  and  general 
signs  of  improvement.  The  treatment  is  continued  with  intermissions 
for  many  months,  and  may  be  resumed,  if  necessary,  from  time  to  time 
over  a  period  of  years. " 

Tuberculin  has  also  been  administered  intrabronchially  and  by  the 
rectum  without  good  results. 

Koch  was  the  first  to  administer  tuberculin  intravenously,  but  this 
route  has  not  come  into  general  favor,  owing  to  the  fact  that  even  greater 
control  over  dosage  is  necessary;  there  is,  besides,  no  local  reaction  to 
serve  as  a  guide,  and  technically  the  administration  is  more  difficult 
than  is  subcutaneous  injection. 

The  Intrafocal  Route. — The  use  of  tuberculin  intrafocally,  that  is,  a 
method  by  which  the  tuberculin  is  brought  into  immediate  contact  with 
the  diseased  area,  has  been  advocated  in  the  treatment  of  tuberculous 
pleurisy,  tuberculous  peritonitis,  and  tuberculosis  of  other  serous  mem- 
branes, such  as  those  lining  joints,  the  tunica  vaginalis  of  the  testicle 
etc.  Senger,  Crocker,  and  Fernet  advise  the  intrafocal  use  of  tuber- 
culin in  the  treatment  of  lupus;  others  have  applied  it  directly  to 
broken-down  glands  and  to  sinuses.  William  Egbert  Robertson1  has 
reported  very  good  results  in  two  cases  of  tuberculous  pleurisy  as  a 
result  of  withdrawing  a  small  amount  of  fluid  and  injecting  5  mg.  of  old 
tuberculin  through  the  needle,  which  is  left  in  situ  for  that  purpose. 
After  some  hours  there  was  a  slight  reaction;  the  remainder  of  the  fluid 
was  rapidly  absorbed,  and  convalescence  was  promptly  established, 
1  Personal  communication. 


730  ACTIVE    IMMUNIZATION 

though,  of  course,  a  damaged  lung  remained.  In  a  case  of  hydrocele  the 
injection  of  old  tuberculin  was  followed  by  a  sharp  local  and  a  moderate 
general  reaction,  followed  by  absorption,  and  without  the  slightest 
evidence  of  recurrence  to  date,  now  about  a  year  since  the  injection  was 
made. 

Results  of  Tuberculin  Therapy. — The  early  and  disastrous  results 
obtained  with  tuberculin  in  various  types  of  tuberculosis  cannot  be  used 
at  the  present  day  as  a  measure  for  determining  the  therapeutic  value  of 
tuberculin. 

So  much  depends  upon  the  individual  case,  the  duration  and  activity 
of  the  disease,  the  possibility  of  supplementing  the  active  immunization 
with  sanatorium  treatment,  the  skill  and  patience  of  the  physician,  etc., 
that,  from  a  prognostic  standpoint,  every  case  must  be  judged  upon  its 
own  merits.  The  results  of  the  modern  use  of  tuberculin  show  quite 
clearly  that  it  is  not  a  "cure"  for  tuberculosis,  but,  rather,  a  rational  and 
useful  therapeutic  aid,  the  best  results  being  secured  when  the  treat- 
ment is  carried  out  in  special  institutions  or  by  specially  trained  physi- 
cians who  have  a  practical  knowledge  of  the  difficulties,  dangers,  and 
possibilities  of  tuberculin  therapy. 

The  value  of  this  or  of  any  therapy  can  be  judged  according  to  various 
standards:  (1)  Working  ability;  (2)  duration  of  life;  (3)  the  presence 
of  tubercle  bacilli  in  the  sputum;  (4)  physical  signs,  and  (5)  the  symp- 
toms. 

The  first  three  are  especially  valuable;  duration  of  life  is,  after  all, 
the  most  important  criterion,  as  anything  that  prolongs  life  is,  of  course, 
welcomed. 

Kremser  chose  110  patients  expectorating  tubercle  bacilli  and  treated 
55  unselected  cases  with  tuberculin;  of  these,  22,  or  40  per  cent.,  lost 
the  bacilli  from  their  sputum;  of  those  treated  without  tuberculin  only 
16,  or  29  per  cent.,  lost  their  bacilli.  Phillippi  found  that  58  per  cent,  of 
his  second  stage  cases  were  rid  of  bacilli  in  the  sputum  under  tuberculin 
treatment,  as  against  19  per  cent,  without.  Brown  reports  from  Saranac 
Lake  that,  in  the  incipient  class,  67  per  cent,  of  the  tuberculin  patients 
were  rid  of  bacilli;  of  the  others,  64  per  cent.  In  the  moderately 
advanced  the  figures  are  respectively  44  per  cent,  and  24  per  cent. 
Bandelier  gives  the  reports  of  500  cases,  of  whom  202  had  tubercle  ba- 
cilli in  the  sputum.  In  the  following  table  he  compares  the  working 
capacity  and  sputum  examinations  of  these  patients  under  tuberculin 
treatment : 


TUBERCULOSIS — TUBERCULIN  THERAPY 


731 


TOTAL 

STAGE  I 

STAGE  II 

STAGE  III 

Complete  earning  capacity 
on  discharge  . 

1 
500  cases  (69.8 

Per  cent. 

Per  cent. 

Per  cent. 

Sputum  changed  from  pos- 
itive to  negative  

per  cent.) 
202  cases  (63.9 

90.4 

80.7 

32.8 

per  cent.) 

100.0 

87.3 

44.0 

The  parallelism  between  the  bacillary  content  of  the  sputum  and  the 
working  capacity  is  close  and  shows  the  value,  from  a  statistical  point 
of  view,  of  sputum  examinations. 

Healing  with  and  without  tuberculin  is  qualitatively  the  same, 
although,  in  the  opinion  of  Ziegler,  Petruschky  and  Rohmer,  Pearson 
and  Gilliland,  Jurgens,  Neumann,  and  others,  quantitatively  the  re- 
sults are  different,  all  authors  agreeing  on  the  presence  of  more  fibrosis 
about  the  lesions  than  is  usual  in  the  untreated  cases.  Autopsy  findings 
necessarily  point  with  less  favor  to  tuberculin  therapy  than  do  clinical 
facts  bearing  on  the  rapidity  and  permanency  with  which  a  lesion  heals. 

The  influence  of  tuberculin  cannot  at  present  be  judged  from  the 
presence  of  antibodies  in  the  serum,  as  data  bearing  upon  the  presence 
or  absence  of  antitoxins,  opsonins,  agglutinins,  and  bacteriolysins  are 
insufficient. 

General  symptoms  and  signs,  such  as  fever,  cough,  loss  of  weight, 
accelerated  pulse,  digestive  disturbances,  dyspnea,  and  pain,  if  they  are 
due  to  tuberculosis,  as  a  rule  improve  under  tuberculin  treatment. 
Under  proper  conditions  the  incidence  of  hemoptysis  is  not  usually 
increased,  and  the  quantity  of  sputum  and  number  of  expectorated 
bacilli  gradually  decrease. 

In  the  treatment  of  tuberculosis  of  the  eye  tuberculin  has  yielded 
exceptionally  good  results;  in  tuberculous  adenitis  considerable  good 
may  be  accomplished  with  those  glands  that  have  not  as  yet  softened. 
In  bone  and  joint  tuberculosis  there  is  a  growing  opinion  that  surgery 
may  be  obviated  or  supplemented  by  tuberculin.  In  tuberculosis  of 
the  ear,  tuberculin  has  yielded  good  results  and  should  always  be  used. 
The  results  in  tuberculosis  of  the  skin  have  been  generally  disappointing, 
and  when  tuberculin  is  administered,  the  treatment  should  be  prolonged 
and  supplemented  by  the  usual  therapeutic  measures.  Tuberculosis  of 
the  intestine  and  mesenteric  glands  may  be  benefited,  and  in  subacute 
tuberculous  meningitis  this  method  of  treatment  may  also  be  tried, 
combined  with  lumbar  puncture  to  relieve  intracranial  pressure.  In 


732  ACTIVE    IMMUNIZATION 

tuberculosis  of  the  genito-urinary  organs  the  prognosis  is  largely  de- 
pendent upon  the  accompanying  pulmonary  condition  and  upon  the 
extent  of  the  local  process.  There  is  general  recognition  of  the  value  of 
tuberculin  as  an  adjuvant  to  hygienic  measures,  although  great  difference 
of  opinion  exists  as  to  surgical  intervention.  If  one  kidney  is  involved, 
most  surgeons  advise  early  operation,  followed  by  tuberculin  treatment; 
if  both  organs  are  diseased,  tuberculin  may  prove  a  valuable  adjuvant 
to  the  treatment. 


CHAPTER  XXX 
PASSIVE  IMMUNIZATION---SERUM  THERAPY 

SERUM  therapy  may  be  said  to  have  had  its  origin  in  1890,  when  von 
Behring  discovered  diphtheria  antitoxin.  He  found  that  guinea-pigs 
surviving  a  subcutaneous  inoculation  of  living  diphtheria  bacilli  may 
harbor  virulent  bacilli  at  the  site  of  injection  without  showing  any 
evidences  of  intoxication.  Subsequent  investigation  showed  that  the 
blood-serum  of  these  animals  contained  the  protective  principles,  for 
when  the  serum  was  injected  into  other  animals  along  with  the  diph- 
theria toxin,  symptoms  of  the  disease  did  not  develop,  and,  indeed,  as 
was  shown  later,  the  immune  serum  was  found  capable  of  neutralizing 
the  toxin  in  the  test-tube.  Shortly  afterward  Kitasato  made  similar 
discoveries  in  studying  tetanus,  and  these  antitoxins  have  since  proved 
of  great  importance,  not  only  from  the  new  light  that  has  been  thrown 
upon  the  mechanism  of  immunity, — and  they  were  used  as  important 
arguments  for  the  humoral  as  opposed  to  the  phagocytic  theory,  and 
form  the  very  basis  and  starting-point  of  Ehrlich's  researches, — but  also 
from  the  new  and  important  field  of  therapy  that  was  now  opened, 
which  gave  promise  and  hope  for  the  discovery  of  a  specific  serum  treat- 
ment for  each  bacterial  disease. 

At  the  time  it  was  thought  possible  to  immunize  animals  with  the 
various  microorganisms  known  to  produce  disease,  and  that  the  immune 
serums  so  produced  may  be  employed  in  the  form  of  specific  treatment. 
This  theory  rested  on  the  fact  that  they  contained  the  antibodies  that 
would  quickly  overcome  the  infection.  With  a  few  of  the  genuinely 
antitoxic  serums  these  hopes  have  been  realized;  but  many  other 
serums  have  not  yielded  the  expected  and  wished-for  results,  although 
at  the  present  time  the  reasons  for  failure  are  being  recognized  and 
gradually  eliminated. 

Definitions. — It  will  be  remembered  that  -in  active  immunization 
our  own  body-cells  are  stimulated  to  produce  antibodies,  either  by 
reason  of  the  presence  of  a  disease  or  as  the  result  of  vaccination  with  the 
antigen  of  the  disease  in  a  modified  and  attenuated  form.  In  passive 
immunization,  however,  our  own  body-cells  do  not  produce  the  antibodies, 

733 


734  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

but  we  receive  them  passively  in  the  form  of  an  injection  of  an  antibody- 
laden  serum.  The  antibodies  are  produced  by  active  immunization  of 
some  other  animal,  usually  a  horse,  and  we  receive  the  antibodies  or  prod- 
ucts of  this  immunization  in  a  passive  manner,  i.  e.,  our  body-cells  receive 
protection  against  an  infection  and  aid  us  in  overcoming  it  through  anti- 
bodies produced  in  some  other  animal.  For  this  reason  the  process  is 
called  passive  immunization;  the  particular  kind  of  increased  resistance 
afforded  against  infection  is  known  as  passive  immunity,  and  since  blood- 
serum  contains  the  antibodies  and  is  the  usual  vehicle  by  which  they  are 
transferred,  the  method  is  called  serum  therapy. 


PURPOSES  OF  PASSIVE  IMMUNIZATION 
Passive  immunization  may  be  employed  for  two  main  purposes: 

1.  To  prevent  disease  (prophylactic  immunization). 

2.  To  cure  disease  (curative  immunization). 

In  prophylactic  immunization  the  antibodies  are  introduced  into  our 
body-fluids  before  infection  has  actually  occurred,  or  at  least  in  the 
earliest  stage  of  infection,  for  the  purpose  of  placing  them  on  guard  to 
destroy  the  infecting  microorganism  or  to  neutralize  its  products  before 
it  has  had  an  opportunity  to  produce  disease.  In  other  words,  we  aim 
to  fortify  our  natural  defenses  by  purchasing  antibodies  from  another 
animal.  From  the  fact  that  these  antibodies  may  be  introduced  in  a 
short  space  of  time  and  that  in  this  manner  an  immunity  may  be  quickly 
gained,  passive  immunization  for  prophylactic  purposes  is  indicated 
when  the  danger  of  infection  is  imminent,  and  when  it  is  impossible,  or 
when  there  is  not  sufficient  time,  for  us  to  stimulate  our  own  body-cells 
to  produce  our  own  antibodies  by  active  immunization  with  a  vaccine. 

Since  the  antibodies  are  produced  in  another  animal,  the  serum,  when 
introduced  into  our  body-fluids,  represents  a  foreign  protein,  and,  ac- 
cordingly, we  find  that  the  antibodies  are  retained  for  relatively  short 
periods  of  time  and  are  quickly  eliminated  or  destroyed.  In  active 
immunization,  however,  the  antibodies  are  in  native  surroundings,  and 
our  body-cells  continue  to  produce  them  for  some  time  after  active 
stimulation  has  ceased,  in  this  manner  insuring  a  higher  degree  of 
immunity  and  one  of  longer  duration.  For  purposes  of  prophylaxis, 
therefore,  active  immunization  is  always  more  desirable  than  passive  im- 
munization; not  infrequently  the  two  forms  are  used  simultaneously, 
as  the  antibody-laden  serum  will  afford  instant  protection,  while  the 
vaccine  is  stimulating  our  body-cells  to  produce  antibodies  that  will 


VARIETIES   OF   PASSIVE    IMMUNITY  735 

increase  and  maintain  the  protection  over  a  longer  period  of  time.  This 
mixed  form  of  immunization  has  recently  received  special  study  by  von 
Behring  in  immunization  experiments  against  diphtheria,  and  will  be 
considered  in  detail  in  a  later  section. 

In  curative  immunization  the  conditions  are  somewhat  different. 
During  the  course  of  an  infectious  disease  our  body-cells  are  actively 
engaged  in  combating  the  infectious  agent,  so  that  reinforcements,  in 
the  form  of  specific  antibodies,  are  indicated  and  welcomed  for  the  aid 
they  give  in  overcoming  an  infection  and  the  relief  they  afford  our  hard- 
pressed  protective  mechanism. 

For  these  reasons  it  may  be  stated  that  the  more  acute  the  infection,  the 
greater  is  the  indication  for  introducing  an  antibody-laden  serum.  In 
chronic  infections  and  in  some  acute  infections  we  may  practise  active 
immunization  by  introducing  a  vaccine,  with  the  purpose  in  mind  of 
stimulating  dormant  cells  to  produce  antibodies;  but,  as  a  rule,  it  is 
reasonable  to  assume  that  in  a  severe  generalized  infection  our  body- 
cells  are  doing  their  utmost  to  overcome  the  infection,  and  extra  stimula- 
tion may  be  actually  harmful.  By  introducing  antibodies  produced  in 
some  other  animal,  however,  practically  no  extra  strain  is  thrown  upon 
the  body-cells;  on  the  contrary,  they  may  be  relieved  when  the  new 
antibodies  overcome  the  products  of  infection,  and  in  this  manner  afford 
them  an  opportunity  to  recover.  Unfortunately,  this  is  actually  the 
case  in  but  a  few  infections,  such  as  diphtheria,  tetanus,  and  cerebro- 
spinal  meningitis,  and  does  not  apply  equally  well  to  the  larger  number 
of  bacterial  infections  due  to  the  pneumococcus,  streptococcus,  gonococ- 
cus,  and  similar  infections.  The  reasons  for  failure  of  passive  immuniza- 
tion in  the  cure  of  the  last-mentioned  infections  are  now  being  studied 
and  realized,  and  means  are  being  discovered  for  overcoming  the  diffi- 
culties, so  that  serum  therapy,  in  a  broad  sense,  is  about  to  play  a  more 
important  role  in  the  treatment  of  many  bacterial  infections. 


VARIETIES  OF  PASSIVE  IMMUNITY 

While,  strictly  speaking,  all  antibodies  are  probably  inimical  to  their 
antigens,  from  the  practical  standpoint  of  passive  immunization  three 
are  of  primary  importance,  namely:  (1)  The  antitoxins,  (2)  the  bac- 
teriolysins,  and  (3)  the  bacteriotropins  (immune  opsonins).  The  anti- 
toxins neutralize  their  toxins;  bacteriolysins  cause  the  death  of  their 
respective  bacteria  if  suitable  complements  are  present,  and  bacterio- 
tropins accomplish  the  same  end  by  lowering  the  resistance  of  the 


736  PASSIVE   IMMUNIZATION — SERUM   THEKAPY 

bacteria,  and  in  this  manner  facilitating  phagocytosis.  Other  antibodies 
may  be  operative  and  prove  of  assistance,  as,  e.g.,  agglutinins  may  aid 
in  bacteriolysis  and  anti-aggressins  may  aid  in  phagocytosis,  but  too 
little  is  known  at  the  present  time  to  allow  fine  distinctions  to  be  made, 
although  the  indications  are  that  not  one  but  several  antibodies  are  present 
in  each  immune  serum,  which,  acting  together,  tend  to  overcome  an  in- 
fection. 

From  the  practical  standpoint,  therefore,  immune  serums  may  be 
used  to  produce  two  main  types  of  passive  immunization,  namely: 

1.  Antitoxic  immunization,  due  to  antitoxins  for  the  true  or  extra- 
cellular toxins,  as  in  diphtheria  and  tetanus  (antitoxic  immunity). 

2.  Antibacterial  immunization,  due  mainly  to  bacteriolysins  and  bac- 
teriotropins,  as  in  meningococcus,  pneumococcus,  streptococcus,  gono- 
coccus,  and  similar  infections  (antibacterial  immunity). 

The  mechanism  of  both  of  these  varieties  of  passive  immunity  will  be 
considered  briefly  under  their  respective  headings. 


INDICATIONS  FOR  PASSIVE  IMMUNIZATION 

For  purposes  of  prophylaxis  only  two  immune  serums  have  proved 
their  efficiency,  namely,  the  antitoxin  of  diphtheria  and  tetanus  anti- 
toxin. 

As  will  be  pointed  out  further  on,  diphtheria  antitoxin,  when  ad- 
ministered in  sufficient  amounts,  affords  protection  for  at  least  from  four 
to  six  weeks;  mixed  immunization,  by  means  of  the  simultaneous  in- 
jection of  a  neutral  mixture  of  the  toxin  and  antitoxin,  as  worked  out  by 
von  Behring,  has  been  found  to  yield  equally  good  and  more  prolonged 
immunity,  but  because  of  certain  technical  difficulties  has  not  as  yet 
been  widely  adopted. 

Tetanus  antitoxin  has  its  greatest  value  as  a  prophylactic.  When 
symptoms  of  tetanus  have  once  appeared,  serum  treatment  may  be  of  no 
avail,  whereas  it  has  proved  its  efficiency  beyond  doubt  in  neutralizing 
the  toxin  before  it  reaches  or  unites  with  the  nervous  tissue.  In  all 
wounds  likely  to  be  infected  with  tetanus  the  physician  should  include 
the  administration  of  tetanus  antitoxin  as  a  matter  of  routine  treatment. 

Of  the  antibacterial  serums,  many  have  a  prophylactic  value  in  ex- 
perimental animals,  but  none,  with  the  exception  of  the  antiplague 
serum,  is  in  general  use  as  a  prophylactic  in  human  practice.  The  rea- 
sons for  this  are  apparent  when  it  is  remembered  that  pneumococcus, 
streptococcus,  and  meningococcus  infections  are  not  sufficiently  epidemic 


INDICATIONS   FOR   PASSIVE    IMMUNIZATION  737 

in  character  to  demand  passive  immunization.  Meningococcus  men- 
ingitis may,  however,  be  an  exception,  but  the  method  of  active  im- 
munization advocated  by  Sophian  is  promising,  easier  to  carry  out,  and 
should  be  tried  during  times  of  epidemic  meningitis. 

In  typhoid  fever,  cholera,  and  dysentery  antibacterial  serums  have 
not  been  generally  used  in  prophylaxis,  although  it  would  appear  that  a 
potent  anticholera  serum  would  prove  of  value  in  preventing  epidemics 
of  this  frightfully  infectious  disease. 

With  the  exception,  therefore,  of  the  true  intoxications,  prophylaxis 
is  more  readily  secured  by  active  than  by  passive  immunization.  This 
is  certainly  true  of  typhoid  fever,  rabies,  and  smallpox.  The  antiserums 
of  other  microorganisms,  such  as  the  pneumococcus,  streptococcus,  and 
gonococcus,  are  being  used  exclusively  for  therapeusis,  rather  than  for 
prophylaxis,  of  their  several  infections. 

In  veterinary  practice  hog-cholera  serum  has  proved  of  value  as  a 
prophylactic  means  of  combating  and  limiting  epidemics  of  hog  cholera. 
It  is  not  definitely  known  whether  this  serum  is  antitoxic  or  antibac- 
terial, but  it  is  probably  a  combination  of  both. 

In  the  treatment  of  disease  immune  serums  have  proved  of  value  in 
diphtheria,  tetanus,  cerebrospinal  meningitis,  and,  to  a  lesser  extent, 
dysentery,  pneumonia,  streptococcus  infections,  and  plague. 

While  antipneumococcus,  antistreptococcus,  and  antigonococcus 
serums  have  proved  of  some  value  in  the  treatment  of  their  particular 
infections,  the  more  recent  work  of  Neufeld  and  Handel,  Dochez  and 
Cole,  and  their  coworkers  in  pneumonia  indicates  that  there  are  wide 
biologic  differences  among  various  strains  of  these  microorganisms,  and 
that  no  curative  properties  can  be  expected  from  a  given  serum  unless 
this  is  homologous  for  the  type  causing  the  infection.  Further  than 
this,  it  has  been  found  impossible  to  secure  serums  as  rich  in  antibodies 
as  are  secured  with  diphtheria  and  tetanus  antitoxins,  and  that  the 
serums  must  be  given  intravenously  in  relatively  large  doses.  A  method 
for  the  quick  recognition  of  types  of  pneumococci  has  been  worked  out 
in  the  Rockefeller  Hospital,  and  immune  serums  have  been  prepared  for 
the  main  types,  and  the  results  of  the  serum  treatment  of  pneumonia 
along  these  lines  have  been  found  to  be  most  encouraging.  While  this 
method  is  not  adapted  for  general  use,  it  holds  out  a  promise  for  the 
future  of  serum  therapy,  and  opens  up  a  wide  field  of  investigation  with 
the  group  of  streptococci,  gonococci,  and  meningococci. 


47 


738  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

CONTRAINDICATIONS  TO  PASSIVE  IMMUNIZATION 

The  chief  contraindications  to  the  therapeutic  use  of  a  serum,  if  any 
ever  exist,  are  those  dependent  upon  the  serum  itself,  for,  as  will  readily  be 
understood,  the  introduction  of  antibodies  themselves  does  not  mean  an 
extra  strain  upon  our  body-cells,  but  rather  the  reverse.  The  question 
before  us,  then,  is  one  regarding  the  possible  contraindications  to  the  in- 
jection of  a  foreign  serum,  and  the  dangers  dependent  upon  its  use.  It 
may  be  stated  at  once  that,  in  the  great  majority  of  cases,  the  adminis- 
tration of  a  carefully  prepared  and  properly  administered  serum  is  free  from 
danger.  Since  the  introduction  of  diphtheria  antitoxin  in  the  prophylaxis 
and  treatment  of  that  disease  many  thousands  of  injections  have  been 
given,  in  all  parts  of  the  world  and  under  all  sorts  of  conditions,  and  the 
number  of  fatalities  is  so  small  as  to  be  regarded  as  almost  negligible. 
Serum  therapy  should  not,  however,  be  abused  to  the  extent  of  using 
the  serum  indiscriminately.  I  am  opposed  to  using  the  serum  as  a 
prophylactic  unless  the  indications  for  its  employment  are  distinct;  for 
example,  in  diphtheria  it  suffices  to  immunize  only  those  who  have  been 
brought  into  immediate  contact  with  the  infection.  When,  however, 
the  indications  are  clear  and  the  symptoms  of  infection  are  present,  I 
believe  in  using  the  serum  early  and  generously. 

1.  Of  all  possible  dangers  consequent  to  the  use  of  serum  therapy, 
that  of  anaphylaxis  is  uppermost  in  the  minds  of  practitioners.  While 
it  is  true  that  anaphylaxis  has  been  the  cause  of  some  fatalities,  the 
likelihood  of  this  accident  taking  place  is  so  remote,  in  the  great  majority 
of  cases,  that  it  should  not  occupy  "a  prominent  place  in  the  physician's 
mind,  nor  interfere  with  the  use  of  the  serum,  as,  for  instance,  anti- 
toxin in  the  treatment  of  diphtheria.  It  is  true  that  serum  sickness  is 
comparatively  common,  and  while  the  symptoms  are  frequently  dis- 
tressing, they  are  not  dangerous  and  do  not  constitute  the  dreaded  and 
fatal  anaphylaxis.  With  a  little  discrimination  and  care  on  the  part 
of  the  physician  the  risk  of  anaphylaxis  may  be  rendered  still  more  re- 
mote if  attention  is  given  to  the  following  questions : 

(a)  Is  the  patient  sensitive  to  horse  protein?  This  is  probably  the 
most  important  single  question,  as  in  several  of  the  fatal  cases  of  ana- 
phylaxis on  record  it  was  learned  afterward  that  the  patient  was  usually 
rendered  uncomfortable,  and  that  sneezing,  asthma,  or  even  an  urti- 
carial  rash  woutd  develop  when  the  patient  came  into  close  proximity  to 
horses,  as  in  a  stable,  or  when  driving  behind  them,  etc.  Fortunately, 
these  cases  are  very  few,  but  several  of  the  fatal  cases  of  anaphylaxis 
on  record  occurred  in  just  such  persons,  and  at  the  present  time  a 


METHODS   OF   INOCULATION  739 

physician  should  generally  be  able  to  detect  this  susceptibility  and 
avoid  the  dangers  of  anaphylaxis.  In  Chapter  XXVIII  the  subject  is 
discussed  in  greater  detail,  and  a  method  of  vaccination  is  described 
by  which  it  may  be  possible  to  detect  this  condition;  this  consists  of 
rubbing  a  little  of  the  serum  into  an  abrasion  on  the  arm  (Fig.  125) .  . 

(6)  Has  the  patient  been  injected  with  a  serum  on  any  former  oc- 
casion? If  an  injection  has  been  given,  especially  a  few  weeks  earlier, 
a  reinjection  of  serum  may  cause  well-marked  serum  sickness,  but  the 
possibilities  of  alarming  anaphylaxis  are  so  remote  that  serum  should 
never  be  withheld  if  the  clinical  condition  indicates  that  it  should  be 
given.  Not  infrequently  a  child  receives  an  immunizing  dose  of  diph- 
theria antitoxin,  but  develops  the  disease  a  month  or  two  later,  after 
the  immunity  has  disappeared.  Under  these  circumstances  antitoxin 
should  not  be  withheld.  If  time  permits,  the  physician  may  inject  0.5 
c.c.  of  the  antitoxin  for  the  purpose  of  producing  anti-anaphylaxis,  fol- 
lowed in  two  or  three  hours  by  the  remainder  of  the  serum.  //  it  were 
possible  to  obtain  it,  it  would  be  good  practice  to  immunize  the  patient  with 
an  ox-serum  antitoxin,  and  then,  if  it  was  found  necessary  later  to  use  an 
antitoxin,  the  usual  horse  serum  antitoxin  could  be  employed.  This  would 
still  further  eliminate  the  possibility  of  the  development  of  disagreeable 
or  dangerous  complications. 

2.  If  a  patient  suffers  from  idiopathic  asthma  and  the  condition 
known  as  status  lymphaticus  develops,  serum  should  be  given  cautiously 
because  of  the  increased  respiratory  difficulties  that  may  follow.     It 
may  be  well  to  give  a  preliminary  hypodermic  injection  of  atropin  and 
caffein,  and  then,  after  a  few  minutes,  give  the  serum,  injected  slowly 
and  subcutaneously. 

3.  Aside  from  these  questions,  the  physician  may  be  called  upon  to 
decide  if  a  patient  is  physically  able  to  withstand  the  effects  of  an  inocu- 
lation, especially  the  intravenous  injection  of  relatively  large  amounts  of 
serumx  such  as  are  given  in  the  treatment  of  pneumonia.     In  diphtheria 
in  very  young  and  weak  children,  when  a  large  number  of  units  or  sev- 
eral injections  are  to  be  given,  concentrated  antitoxin  is  to  be  preferred, 
in  order  that  injury  to  the  subcutaneous  tissues,  pain,  and  shock  may  be 
reduced  to  a  minimum. 


METHODS  OF  INOCULATION 

Upon  the  nature  and  severity  of  the  infection  will  depend  the  question 
whether  the  serums  are  to  be  given  subcutaneously,  intramuscularly, 


740  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

intravenously,  or  intraspinously  (subdurally).  In  diphtheria,  the  anti- 
toxin may  be  given  subcutaneously  unless  the  infection  is  quite  severe; 
in  the  latter  case  it  should  be  given  intramuscularly  or  intravenously. 
In  tetanus  the  serum  should  be  given  subdurally  and  intravenously. 
In  epidemic  cerebrospinal  meningitis  the  serum  is  always  given  sub- 
durally. In  pneumococcus,  streptococcus,  and  gonococcus  infections, 
while  the  serum  may  be  given  subcutaneously  or  intramuscularly,  it  is 
best  administered  intravenously.  It  is  important  for  the  physician  to 
know  and  appreciate  that  the  route  and  method  of  inoculation  and  the 
amount  of  serum  administered  are  important  factors  in  determining  the 
success  or  failure  of  serum  therapy. 

f.  TECHNIC  OF  SUBCUTANEOUS  INOCULATION 

Serum  given  subcutaneously  is  slowly  absorbed,  and  a  portion  of  the 
antibodies  may  be  destroyed  before  they  reach  the  blood-stream.  When 
large  quantities  of  serum  are  to  be  given,  as  in  pneumonia  and  strepto- 
coccus infections,  this  method  may  not  be  permissible  on  account  of  the 
pain  and  injury  to  the  subcutaneous  tissues  that  may  result,  aside  from 
the  more  important  question  of  slow  absorption  and  anchorage  or  de- 
struction of  the  antibodies  in  various  tissues  before  they  reach  the  blood- 
stream or  the  focus  of  disease. 

1.  Injections  should  be  given  where  the  subcutaneous  tissues  are 
loose,  where  movement  is  least  marked,  and  preferably  where  pressure 
upon  the  parts  is  least  likely  to  occur,  for  some  soreness,  dependent  upon 
the  bulk  of  the  injection,  is  bound  to  follow.     For  these  reasons  injec- 
tions may  be  given  in  the  abdominal  wall;   some  prefer  the  back,  in  the 
region  of  the  lower  angle  of  one  of  the  scapula,  and  the  buttocks,  but  in  a 
bedfast  patient  pressure  at  these  points  cannot  readily  be  eliminated. 

2.  The  skin  about  the  site  for  injection  may  be  prepared  by  an  ap- 
plication of  tincture  of  iodin;  this  is  washed  off  with  alcohol  just  before 
the  needle  is  inserted.     After  the  injection  has  been  given  the  remaining 
iodin  should  be  removed  with  alcohol,  to  prevent  the  occurrence  of  a 
dermatitis,  and  the  puncture  wound  covered  with  cotton  and  collodion 
or  with  sterile  gauze  fastened  with  adhesive  straps. 

3.  The  syringe  and  needle  should  be  sterile.     Manufacturers  of  bio- 
logic supplies  furnish  antitoxin  in  syringes  ready  for  injection,  and  these 
are  usually  convenient  and  satisfactory.     The  needle  should  be  of 
medium  size,  and  larger  than  that  used  for  ordinary  hypodermic  medica- 
tion.    All  glass  or  glass  and  metal  syringes  that  may  be  boiled  are  to  be 
preferred  when  a  syringe  is  not  furnished.     Before  boiling  such  a  syringe 


METHODS    OF   INOCULATION 


741 


the  piston  should  be  removed  from  the  barrel,  as  otherwise  it  may  expand  so 
rapidly  as  to  cause  the  latter  to  crack. 

4.  When  all  is  in  readiness,  the  syringe  being  loaded  and  the  air  ex- 
pelled, the  skin  is  pinched  up  between  the  fingers  and  the  needle  quickly 
inserted  into  the  subcutaneous  tissues.  The  injection  should  be  given 
slowly,  and  during  the. operation,  if  the  patient  is  a  child,  an  assistant 
should  be  on  hand  to  prevent  struggling.  In  the  illustration  (Fig.  138) 
the  needle  is  shown  connected  with  the  barrel  of  the  syringe  by  means  of  a 


FIG.  138. — SUBCUTANEOUS  INJECTION  OF  SERUM. 

The  site  of  injection  is  painted  with  tincture  of  iodin  and  covered  with  sterile 
gauze  fastened  with  straps  of  adhesive  plaster.  Just  before  the  injection  is  given 
the  iodin  is  wiped  off  with  a  pledget  of  cotton  and  alcohol.  A  fold  of  skin  is  pinched 
up  between  the  thumb  and  forefinger  of  the  left  hand,  the  needle  inserted,  and  the 
serum  slowly  injected.  The  needle  is  then  quickly  withdrawn,  and  the  puncture 
covered  with  the  gauze  and  held  in  place  by  the  adhesive  plaster. 


short  piece  of  rubber  tubing.  This  permits  an  injection  to  be  given 
without  danger  of  the  needle  being  broken  off  if  the  patient  should 
struggle.  Most  pain  is  experienced  when  the  first  few  drops  of  fluid  are 
injected;  after  that  the  pain  is  not  severe  unless  the  tissues  are  suddenly 
distended,  as  by  a  quick  injection. 

The  amount  of  serum  that  may  be  injected  in  one  area  depends  upon 
the  age  of  the  patient.  Due  care  should  be  exercised  against  injecting 
too  much  serum  in  one  area,  because  of  slower  absorption  and  possible 
necrosis  of  the  skin  and  subcutaneous  tissues. 


742  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

2*  TECHNIC  OF  INTRAMUSCULAR  INOCULATION 

As  shown  experimentally  by  Meltzer  and  Auer,  absorption  occurs 
much  more  quickly  when  inoculations  are  given  into  the  muscles  than 
when  they  are  given  into  the  subcutaneous  tissues.  For  this  reason  anti- 
toxin should  be  given  intramuscularly  in  severe  cases  of  diphtheria,  as 
the  technic  is  just  as  simple  as  that  of  a  subcutaneous  injection.  When- 
ever the  physician  desires  more  speedy  absorption  than  that  which  fol- 
lows a  subcutaneous  injection,  and  the  intravenous  route  cannot,  for 
some  reason,  be  adopted,  the  inoculation  should  be  given  in  the  muscles, 
preferably  those  of  the  buttocks. 

The  technic  is  the  same  as  that  employed  for  subcutaneous  injections, 
except  that  the  needle  is  plunged  deeply  into  the  muscles.  If  the  most 
muscular  portions  are  selected  for  injection,  there  will  be  little  or  no 
danger  of  injuring  a  nerve.  If  desired,  the  syringe  may  be  detached 
after  the  needle  has  been  inserted  to  ascertain  if  a  vein  has  been  entered, 
which  would  be  shown  by  a  flow  of  blood.  If  this  occurs,  the  needle 
should  be  withdrawn  slightly  and  passed  in  another  direction. 

3.  TECHNIC  OF  INTRAVENOUS  INOCULATION 

The  necessity  of  administering  serum  intravenously  in  order  to 
obtain  the  best  results,  or  any  result  at  all,  is  becoming  more  and 
more  apparent.  In  severe  cases  of  diphtheria  the  best  results  are 
obtained  when  the  antitoxin  is  given  intravenously;  in  the  treatment 
of  tetanus  the  tetanus  antitoxin  should  be  administered  intra- 
venously as  well  as  intradurally,  and  both  antistreptococcus  and  anti- 
pneumococcus  serums  should  always  be  given  by  the  intravenous  route. 
Recent  reports  indicate  that  the  proper  serum  treatment  of  these  in- 
fections requires  large  doses  given  intravenously.  Physicians  should, 
therefore,  be  prepared  to  give  intravenous  injections.  Since  the  use  of 
salvarsan  in  the  treatment  of  syphilis  has  become  so  popular  many 
workers  have  perfected  themselves  in  the  technic  of  intravenous  adminis- 
tration, but  there  is  still  great  hesitancy  about  giving  intravenous  in- 
jections, although  the  methods  are  relatively  simple  and  easily  mastered. 
With  nervous  patients  the  injections  can  be  made  practically  painless 
by  the  preliminary  infiltration  of  the  skin  about  the  site  of  puncture 
with  a  few  drops  of  a  sterile  1  per  cent,  solution  of  eucain. 

Syringe  Method. — When  small  amounts  of  fluid  are  to  be  injected, 
as  from  5  to  20  c.c.,  a  syringe  is  employed. 

1.  It  is  best  to  use  an  all-glass  syringe,  or  at  least  one  with  a  glass 
barrel,  for  the  physician  can  then  assure  himself  that  all  air  has  been  ex- 
pelled  and  that  the  fluid  is  free  from  solid  particles.  Further  than  this,  a 


METHODS   OF   INOCULATION  743 

flow  of  blood  into  the  syringe  will  indicate  that  the  needle  has  entered 
the  vein.  The  syringes  furnished  by  manufacturing  firms  are  not 
well  adapted  for  making  these  injections,  as  the  rubber  plunger  fre- 
quently adheres  to  the  glass  barrel,  so  that  the  injection  will  be  jerky 
and  difficult,  and,  besides,  it  may  be  difficult  to  determine  when  the  vein 
has  been  entered.  It  is  better  to  empty  the  contents  of  these  syringes 
into  a  large,  sterilized,  glass-barreled  syringe,  such  as  the  Record,  Luer, 
and  Burroughs-Wellcome  syringes,  which  have  a  close-fitting  but  easily 
working  piston,  and  are  attached  to  the  needle  by  a  flange  and  not  by  a 
screw  thread.  (See  Fig.  8.)  The  needle  should  be  sufficiently  large 
and  have  a  sharp  but  short  beveled  edge.  A  long  point  may  pierce  the 
vein  through  and  through,  and  permit  perivascular  bleeding  or  result 
in  a  subcutaneous  injection. 

2.  In  young  children  with  fat  arms  and  a  weak  circulation  it  is  usu- 
ally necessary  to  expose  a  vein  at  the  elbow  by  making  a  small  incision. 
In  older  children  and  adults  a  vein  may  stand  out  prominently  enough 
to  permit  the  needle  to  be  inserted  directly  through  the  skin  without 
making  an  incision.     A  firm  rubber  tourniquet  is  applied  above  the 
elbow;  a  very  simple  one  is  constructed  by  a  single  turn  around  the  arm 
with  a  piece  of  ordinary  soft-rubber  tubing  held  in  place  by  a  hemostat. 
After  the  vein  has  been  entered  the  tourniquet  should  be  quickly  removed 
and  this  is  quickly  and  deftly  accomplished  by  releasing  the  hemostat. 

3.  The  skin  about  the  site  of  injection  is  cleansed  with  soap,  water, 
and  alcohol,  or  merely  painted  with  iodin,  which  is  removed  with  alcohol 
just  before  the  injection  is  to  be  given,  in  order  that  the  vein  may  become 
visible. 

4.  An  assistant  steadies  the  patient's  arm  and  should  be  ready  to 
release  the  tourniquet. 

5.  The  operator  then  steadies  the  skin  over  a  vein — usually  the 
median  basilic  or  median  cephalic — with  the  left  thumb  and  forefinger, 
and  introduces  the  needle  into  the  vein.     A  flow  of  blood  into  the  syringe 
indicates  that  the  vein  has  been  entered.     The  tourniquet  is  then  released 
and  the  injection  slowly  given.     Or  the  needle  may  be  detached  from 
the  syringe  and  passed  into  the  vein;   when  blood  appears,  the  syringe 
is  quickly  attached  and  the  injection  made.     The  puncture  wound  is 
then  sealed  with  a  wisp  of  sterile  cotton  and  collodion  or  with  gauze  and 
a  bandage.     The  syringe  shown  in  Fig.  139  is  well  adapted  for  the  in- 
travenous injection  of  serum,  and  was  devised  for  the  administration  of 
concentrated  solutions  of  salvarsan  and  neosalvarsan,  but  any  reliable 
and  large  glass-barreled  syringe  may  be  used. 

Gravity  Method. — Larger  quantities  of  serum  or  other  fluid  are 


744 


PASSIVE   IMMUNIZATION — SERUM    THERAPY 


better  injected  by  the  gravity  method,  the  simple  apparatus  shown  in 
Fig.  140  being  quite  satisfactory  for  the  purpose.  This  consists  merely 
of  a  graduated  cylinder,  which  serves  as  a  mea  uring  funnel,  rubber  tub- 
ing with  a  pinch  cock,  and  is  furnished  with  a  metal  tip  that  fits  the 
needle.  The  needle  should  be  of  proper  size,  and  have  a  sharp  but  some- 
what short  beveled  edge.  It  may  be  curved,  as  shown  in  the  illustration, 
or  may  be  straight.  The  apparatus  shown  in  Fig.  146  is  adapted 


FIG.  139. — METHOD  OF  MAKING  INTRAVENOUS  INJECTION  BY  MEANS  OF  A  SYRINGE. 
This  syringe  was  devised  for  the  intravenous  administration  of  a  concentrated 
solution  of  salvarsan.  It  is  provided  with  a  three-way  cock,  which  permits  drawing 
fluid  into  the  syringe  and  then  injecting  it  into  a  vein.  This  injection  may  also  be 
given  by  any  glass  syringe;  the  particular  advantage  of  this  one  is  that  the  opera- 
tor may  inject  more  than  one  syringeful  of  fluid  without  removing  the  needle.  The 
same  syringe  may  be  used  for  the  intravenous  injection  of  any  serum,  as  diphtheria 
and  tetanus  antitoxins.  (Apparatus  made  by  B.  B.  Cassel,  Frankfort,  Germany.) 

for  the  administration  of  salvarsan,  and  has  the  decided  advantage  of 
permitting  the  operator  to  give  an  intravenous  injection  to  an  adult 
person  without  assistance. 

1.  The  cylinder,  tubing,  and  needle  should  be  sterilized  by  boiling 
prior  to  use. 

2.  The  serum  or  other  fluid  may  be  warmed  by  placing  the  container 
in  water  of  a  temperature  not  higher  than  42°  C.  (just  comfortably  hot 
to  hold  the  hand  in). 


METHODS    OF   INOCULATION 


745 


3.  The  injections  are  best  given  in  a  vein  at  the  elbow.  The  arm 
about  this  region  should  be  scrubbed  with  hot  water  and  soap,  followed 
by  alcohol  and  1 : 1000  bichlorid  of  mercury  solution,  or  liberally  painted 
with  tincture  of  iodin.  A  firm  tourniquet  is  then  applied  above  the 
elbow;  a  single  firm  turn  of  rubber  tubing  held  by  a  hemostat  is  quite  sat- 
isfactory, as  when  the  vein  has  been  entered  the  tourniquet  should  be 
quickly  released  with  the  least  movement  and  disturbance  possible,  and 


FIG.  140. — METHOD  OF  MAKING  INTRAVENOUS  INJECTION  BY  GRAVITY. 

This  method  is  suitable  for  the  intravenous  administration  of  salvarsan  or  anti- 
streptococcus  serum,  etc.  The  needle  has  been  entered  into  a  prominent  vein 
(indicated  by  a  flow  of  blood);  the  tubing  has  been  attached  by  means  of  a  metal 
tip  which  fits  the  needle  easily  and  snugly;  the  tourniquet  has  been  loosened  and  the 
injection  is  being  given. 

this  arrangement  answers  all  requirements.     Sterile  towels  should  be 
placed  about  the  arm  and  shoulder. 

4.  About  20  c.c.  or  more  of  sterile  distilled  water  or  normal  salt  solu- 
tion are  then  poured  into  the  cylinder,  and  the  cock  opened  until  all  air 
has  been  expelled  from  the  tubing.  The  fluid,  serum,  or  salvarsan  is 
then  poured  into  the  cylinder.  It  is  a  good  practice  to  filter  the  fluid 
through  several  layers  of  sterile  gauze,  especially  when  salvarsan  is  being 
injected,  in  order  to  remove  any  bits  of  glass  or  other  foreign  bodies  that  may 
be  present. 


746  PASSIVE   IMMUNIZATION — SERUM    THERAPY 

5.  An  assistant  holds  the  loaded  cylinder  and  tubing;  the  operator 
steadies  the  skin  over  a  prominent  vein  and  quickly  inserts  the  needle. 
A  flow  of  blood  indicates  that  the  vein  has  been  penetrated.  The  tub- 
ing is  then  quickly  and  carefully  attached,  the  tourniquet  released  by 
unfastening  the  hemostat,  and  the  injection  given.  As  a  rule,  an  eleva- 
tion of  the  cylinder  of  two  or  three  feet  is  sufficient.  If  swelling  occurs 
about  the  site  of  puncture  and  the  patient  complains  of  pain,  the  in- 
jection is  entering  the  subcutaneous  tissue;  when  this  occurs,  the  pinch 
cock  should  be  closed  and  the  needle  removed.  It  is  then  necessary  to 
make  the  injection  into  another  vein  or  into  the  same  vein  at  another 
site. 

TECHNIC  OF  SUBDURAL  INOCULATION 

In  the  treatment  of  epidemic  cerebrospinal  meningitis,  influenzal 
meningitis,  and  tetanus  the  specific  serums  are  administered  subdurally 
by  means  of  a  needle  introduced  in  the  lumbar  region.  Recently  sub- 
dural  injections  of  salvarsanized  serum  and  weak  solutions  of  salvarsan 
itself  have  been  advocated  in  the  treatment  of  cerebrospinal  syphilis, 
tabes  dorsalis,  and  paresis.  Every  practitioner  should  be  prepared  to 
perform  lumbar  puncture  for  the  purpose  of  securing  cerebrospinal  fluid 
for  making  the  Wassermann  reaction  and  the  bacteriologic,  cytologic, 
and  chemical  examinations,  and  the  administration  of  serum  is  a  rela- 
tively simple  matter  when  the  puncture  has  been  successfully  made. 

The  technic  of  lumbar  puncture  for  the  purpose  of  securing  fluid  for 
diagnosis  is  described  on  p.  37.  But  when  administering  serum,  and 
especially  in  the  treatment  of  meningitis,  the  clinical  condition  of  the 
patient  and  the  danger  of  sudden  collapse  render  it  advisable  and  neces- 
sary that  the  inoculation  be  given  with  the  patient  lying  on  his  side. 

Methods. — Two  methods  are  now  being  employed.  The  older  method 
consists  in  injecting  the  serum  by  means  of  a  syringe,  and  the  later  one 
is  a  method  whereby  the  serum  is  allowed  to  flow  in  by  gravity. 

Not  infrequently  a  patient  will  develop  symptoms  of  collapse  during 
a  subdural  injection,  and  these  have  been  ascribed  to  undue  pressure, 
the  injurious  action  of  trikresol  or  other  preservative  upon  the  respira- 
tory centers,  too  rapid  injection,  and  the  introduction  of  too  large  a 
quantity  of  serum.  It  is  now  apparent  that  in  the  past  too  little  atten- 
tion has  been  paid  to  the  patient  while  the  injection  was  being  made,  and 
serum  has  usually  been  administered  according  to  more  or  less  fixed  and 
arbitrary  rules,  instead  of  being  guided  by  the  clinical  condition  of  the 
patient. 


METHODS   OF   INOCULATION  747 

If  symptoms  of  collapse  appear  during  a  subdural  injection,  they  may 
be  relieved  by  allowing  the  fluid  within  the  canal  to  flow  out  again,  and 
this  is  best  accomplished  when  the  inoculation  is  given  by  the  gravity 
method.  The  latter  method  has  been  largely  worked  out  and  is  highly 
recommended  by  Sophian,  these  recommendations  being  based  upon 
his  extensive  experience  in  the  recent  Texas  epidemic  of  cerebrospinal 
meningitis.  It  is  also  recommended  by  Flexner  and  the  Hygienic 
Laboratory,  and  is  undoubtedly  the  method  of  choice. 

Blood-pressure  as  a  Guide  in  Administering  Serum  Subdurally. — 
According  to  the  older  and  customary  method  of  injecting  serum  sub- 
durally,  fluid  is  permitted  to  flow  from  the  needle  until  from  15  to  20 
c.c.  have  been  removed,  and  an  equal  quantity  of  serum  is  then  injected. 
In  severe  cases,  with  thick  plastic  exudates,  only  a  few  cubic  centimeters 
of  fluid  may  be  withdrawn,  and,  indeed,  no  fluid  at  all  may  be  secured. 
To  inject  arbitrarily  a  fixed  amount  of  serum  under  such  conditions  may 
be  highly  dangerous  to  the  patient,  on  account  of  increased  pressure. 
On  the  other  hand,  when  the  flow  is  free,  it  may  be  dangerous  to  permit 
the  canal  to  drain  until  intraspinal  pressure  is  reduced  to  the  normal,  a 
fact  indicated  by  the  flow  of  a  drop  of  fluid  every  three  to  five  seconds. 

With  these  considerations  in  mind,  Sophian1  has  studied  the  value 
of  cerebrospinal  fluid  pressure  and  blood-pressure  as  controls-  on  the 
amount  of  fluid  that  may  be  safely  withdrawn  and  on  the  amount  of 
serum  that  may  be  injected.  During  the  study  and  treatment  of  500 
cases  of  epidemic  cerebrospinal  meningitis  this  last-named  observer 
found  that  the  blood-pressure  was  a  valuable  guide.  Cerebrospinal 
fluid  pressure  was  found  to  be  misleading,  owing  probably  to  a  local  dis- 
tention  of  the  subarachnoid  space  at  the  site  of  injection,  which  resulted 
in  readings  that  did  not  represent  the  true  intracranial  pressure. 

1.  Usually,  upon  the  withdrawal  of  cerebrospinal  fluid,  a  fall  of 
blood-pressure  occurs.     With  the  ordinary  blood-pressure  in  an  adult 
patient — about  110  mm.  of  mercury — Sophian  recommends  stopping 
the  flow  when  there  has  been  a  drop  in  pressure  of  about  10  mm.  of 
mercury;   in  children,  .about  5  mm.     In  a  few  cases  there  is  no  change 
in  blood-pressure  or  even  a  slight  rise;    in  these  instances  fluid  may  be 
removed  until  the  flow  has  diminished  to  the  rate  of  a  drop  every  three 
to  five  seconds. 

2.  With  the  injection  of  serum  the  blood-pressure  drops  still  further. 
Generally,  the  decrease  in  blood-pressure  is  proportional  to  the  rapidity 
with  which  the  serum  is  injected  and  the  amount  injected.     By  the 

1  Epidemic  Cerebrospinal  Meningitis,  1913,  Mosby  Co.,  St.  Louis. 


748  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

gravity  method,  under  ordinary  conditions,  at  least  ten  minutes  should 
be  consumed  in  administering  15  c.c.  of  serum.  A  total  drop  of  20  mm. 
of  mercury  indicates  that  sufficient  serum  has  been  injected.  If  it  is  de- 
sired to  inject  more,  as  in  a  severe  case  of  meningitis,  close  watch  should 
be  kept  for  other  symptoms  of  collapse. 

3.  Usually,  under  these  conditions,  less  serum  is  administered  than 
has  been  advocated  heretofore.     It  is  apparent  that  the  more  potent 
the  serum,  the  less  bulk  is  required — and  the  bulk  alone  is  an  important 
factor,  for  a  large  injection  may  so  injure  the  patient  as  to  counteract 
any  good  that  the  serum  may  do.     Unfortunately,  there  is  no  accurate 
measure  of  the  curative  value  of  antimeningococcic  serum.     It  is  highly 
desirable  that  a  serum  be  as  potent  as  possible,  and  the  physician  must 
rely  upon  the  reputation  of  the  firm  producing  the  serum.     Efforts  are 
being  made  to  concentrate  these  serums,  much  as  antitoxin  is  concen- 
trated, and  this  is  an  end  very  much  to  be  desired. 

4.  Blood-pressure  changes  are  not  constant  in  the  same  patient  upon 
different  occasions.     The  pressure  should  be  taken,  after  each  puncture 
and  inoculation,  for  the  administration  cannot  be  guided  by  observa- 
tions made  on  a  previous  occasion. 

Collapse  during  Subdural  Inoculation. — Carter  has  shown,  by  ex- 
periments on  dogs,  that  the  first  mechanical  effects  of  increased  intra- 
spinal  pressure  were  respiratory  depression  and  marked  cardiac  inhibi- 
tion. Sophian  has  found  that  similar  effects  may  be  produced  during 
subdural  injections  of  serum  in  the  treatment  of  meningitis. 

The  symptoms  of  collapse,  such  as  stupor,  superficial  or  deep,  ir- 
regular and  slow  respiration,  and  dilatation  of  the  pupils,  are  fore- 
shadowed by  a  marked  drop  in  blood-pressure.  The  pulse  may  con- 
tinue good  or  become  slow  and  irregular.  Incontinence  of  urine  and 
feces  may  occur. 

The  treatment  consists  primarily  in  discontinuing  the  injection.  By 
lowering  the  funnel,  fluid  is  allowed  to  flow  from  the  spinal  canal  and 
mix  with  the  serum.  If  a  syringe  is  being  used,  it  should  be  detached 
from  the  needle  or  gentle  suction  made.  After  a  few  minutes  the  symp- 
toms may  disappear  and  the  inoculation  may  be  cautiously  resumed 
until  the  desired  amount  of  serum  has  been  injected;  otherwise  the 
needle  should  be  withdrawn. 

In  addition  to  this  procedure  atropin  and  caffein  may  be  administered 
hypodermically  in  large  doses,  and  artificial  respiration  resorted  to  if 
necessary.  It  is  well  to  have  these  drugs  ready  for  injection  before  the 
inoculation  is  begun,  so  that  no  time  will  be  lost  when  they  are  needed. 


METHODS   OF   INOCULATION  749 

Anesthesia  for  Subdural  Inoculation. — There  is  no  doubt  but  that 
lumbar  puncture  and  the  subdural  injection  of  fluid  are  painful,  the 
amount  of  pain  depending  to  some  extent  upon  the  degree  of  meningitis, 
the  method  of  injection,  and  the  skill  of  the  operator.  Severe  cases  of 
meningitis  that  are  stuporous  or  moribund  may  not  evince  any  evidences 
of  added  discomfort;  less  toxic  and  robust  or  nervous  patients  may,  how- 
ever, suffer  considerably  and  prove  difficult  subjects  for  injection. 

Local  anesthesia  may  be  secured  by  injecting  a  sterile  1  per  cent, 
solution  of  eucain  in  the  region  where  the  puncture  is  to  be  made.  Gen- 
eral anesthesia  for  lumbar  puncture  in  meningitis  adds  a  considerable 
element  of  danger,  but  if  it  is  absolutely  necessary,  a  few  whiffs  of  ether 
or  chloroform  may  be  given  while  the  needle  is  being  inserted.  In  giving 
subdural  injections  in  tetanus  a  general  anesthetic  is  necessary. 

Sophian  has  found  that  if  water  is  given  through  a  straw  while  per- 
forming lumbar  puncture  patients  will  frequently  drink  Jarge  quantities 
of  it  and  keep  very  quiet. 

Gravity  Method. — The  apparatus  required  is  very  simple,  and  con- 
sists essentially  of  a  proper  needle  and  from  12  to  16  inches  of  soft- 
rubber  tubing  attached  to  a  container  or  funnel  for  serum  and  furnished 
with  a  metal  tip  by  which  it  is  quickly  and  readily  attached  to  the 
needle. 

Several  manufacturers  of  biologic  supplies  are  marketing  antimenin- 
gococcic  serum  in  a  special  container,  fashioned  after  that  devised  by 
Sophian  and  Alexander,  with  the  needle  and  tubing  adapted  for  the 
administration  of  the  serum  by  the  gravity  method.  Such  an  apparatus 
is  shown  in  Fig.  141. 

The  physician  may,  however,  prepare  an  equally  efficient  apparatus, 
similar  to  that  shown  in  Fig.  14  i,  which  consists  of  the  glass  barrel  of  a 
20  c.c.  syringe  attached  to  16  inches  of  soft-rubber  tubing  fitted  with  a 
metal  tip  that  holds  the  needle  firmly  and  snugly.  The  whole  is  steril- 
ized by  boiling,  and  any  quantity  of  serum  may  be  administered  with  it. 
The  apparatus  is  adapted  for  the  administration  of  antimeningococcic 
serum,  tetanus  antitoxin,  influenza  serum,  salvarsanized  serum,  or  any 
other  fluid,  and  has  given  uniform  satisfaction. 

The  needle  should  be  from  10  to  11  cm.  in  length,  with  a  wide,  rather 
than  a  narrow,  lumen — about  1.5  to  2  mm.  This  is  important  in  ad- 
ministering serum  to  a  case  of  meningitis,  in  which  a  needle  with  a  nar- 
row lumen  may  become  plugged  with  exudate.  The  needle  should  be 
fitted  with  a  trocar.  The  tip  should  have  a  short  bevel  with  a  sharp 
edge. 


750 


PASSIVE   IMMUNIZATION — SERUM    THERAPY 


Technic. — 1.  The  serum  should  be  warmed  to  body  temperature  by 
wrapping  the  sealed  container  of  serum  in  towels  wrung  out  of  water 
comfortably  hot  for  the  hands  (about  42°  C.).  Cold  serum  possibly 
increases  the  pain  caused  by  the  injection,  although  the  pressure  upon 


FIG.  141. — OUTFIT  FOR  INTRASPINAL  INJECTION  OF   ANTIMENINGITIS  SERUM  BY 

GRAVITY  (Sophian). 

the  sensitive  nerve-roots  is  the  chief  source  of  pain  and  discomfort.  Due 
care  must  be  exercised  that  the  serum  is  not  coagulated  by  too  high 
temperature. 

2.  The  funnel  or  syringe  barrel,  tubing,  and  needle  should  be  steril- 
ized by  boiling.     The  outfits  furnished  by  the  manufacturers  are  steril- 


METHODS    OF    INOCULATION 


751 


ized  and  ready  for  use.  Two  sterile  graduated  centrifuge  tubes  should 
be  on  hand  for  collecting  and  measuring  the  spinal  fluid.  The  apparatus 
should  be  assembled  and  ready  for  injection,  so  that  at  the  appointed 
time  the  tubing  may  be  attached  to  the  needle  and  the  inoculation  given. 
3.  The  patient  should  be  placed  on  the  left  side,  on  the  edge  of  a  bed 
or  table.  An  assistant  places  the  patient  in  such  a  manner  as  to  arch 


FIG.  142. — INTRASPINAL  INJECTION  BY  GRAVITY. 

The  line  marks  the  crest  of  the  ilium,  and  indicates  the  third  lumbar  interspace. 
The  needle  has  been  inserted  and  cerebrospinal  fluid  withdrawn.  Antiminingococcus 
serum  is  being  administered.  The  barrel  of  a  20  c.c.  Record  syringe  is  serving  as  a 
funnel,  and  is  attached  to  the  needle  by  means  of  18  inches  of  soft-rubber  tubing 
furnished  with  a  metal  tip.  The  needle,  as  shown,  is  reduced  to  about  four  times  its 
actual  size.  This  method  may  be  used  for  making  the  subdural  injection  of  tetanus 
antitoxin  and  salvarsanized  serum. 


the  back  as  much  as  possible.     A  second  assistant  takes  blood-pressure 
readings  during  the  operation. 

4.  Towels  wet  with  bichlorid  are  arranged  about  the  site  of  inocula- 
tion. It  is  well  for  the  operator  to  locate  the  site  of  injection  by  pal- 
pating the  spinous  processes  and  selecting  the  widest  interspace,  which 
is  usually  on  a  level  with  the  crests  of  the  ilia  if  the  back  is  well  arched. 
The  skin  is  then  cleansed  with  soap,  water,  and  alcohol,  and  bichlorid 
solution  or  a  coat  of  iodin  applied.  Some  degree  of  local  anesthesia  may 
be  secured  by  injecting  a  small  amount  of  a  sterile  1  per  cent,  solution 
of  eucain.  This  is  advisable  in  nervous  adults. 


752  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

5.  The  operator  must  then  choose  between  the  median  or  lateral 
route  of  puncture.     The  median  is  the  easier,  and  should  always  be 
adopted  by  the  inexperienced  operator. 

Wash  off  the  iodin  with  a  pledget  of  cotton  soaked  in  alcohol.  Lo- 
cate the  chosen  interspinous  space,  pressing  well  between  the  spines 
with  the  left  thumb  or  index-finger,  and  holding  the  finger  in  place  pass 
the  needle  perpendicularly  in  the  median  line  between  the  spines,  or, 
better  still,  at  an  angle  of  45  degrees  upward  and  inward.  If  an  ob- 
struction is  felt,  withdraw  the  needle  slightly  and  pass  it  in  a  different 
direction  until  it  imparts  a  sense  of  " giving  way,"  which  indicates  that 
the  subarachnoid  space  has  been  reached.  Quincke  has  estimated  the 
depth  of  lumbar  puncture  in  adults  to  be  usually  from  4  to  6  cm. ;  in 
large  muscular  men  it  is  from  7  to  8  cm.,  and  in  fat  persons,  about  10 
cm. 

The  needle  should  be  inserted  slowly  and  deliberately,  rather  than 
quickly,  as  puncture  of  a  bone  is  likely  to  be  followed  by  a  dull,  aching 
pain,  and,  indeed,  the  point  of  the  needle  may  be  bent  or  broken. 

The  fluid  may  fail  to  flow  or  flow  very  slowly.  This  may  be  due  to 
the  presence  of  a  thick  exudate,  impalement  of  a  nerve  filament,  or  ad- 
hesions arising  from  a  previous  puncture.  The  needle  may  be  turned 
gently  or  the  trocar  inserted  to  remove  an  obstruction,  after  which  the 
flow  usually  starts;  if  it  does  not  do  so,  the  needle  may  be  withdrawn 
slightly  or  cautiously  inserted  a  little  further. 

6.  Fluid  is  collected  in  the  centrifuge  tubes  while  blood-pressure 
readings  are  being  made.     When  the  pressure  drops  10  mm.,  or  if  the 
flow  is  about  a  drop  every  three  or  five  seconds,  the  tubing  is  connected 
and  the  serum  injected  very  slowly. 

As  a  general  rule,  as  much  fluid  should  be  withdrawn  as  can  be  done 
with  safety,  and  the  maximum  dose  of  serum  given.  When  the  flow  is 
scanty,  a  larger  dose  of  serum  may  be  given  than  counterbalances  the 
fluid  removed,  the  injection  being  guided  by  the  blood-pressure.  When 
the  total  drop  reaches  20  mm.  of  mercury,  the  injection  should  be  dis- 
continued, or  if  continued,  the  patient  should  be  watched  closely  for 
other  symptoms  of  collapse. 

7.  After  the  injection  has  been  completed  the  needle  is  quickly  with- 
drawn and  the  wound  covered  with  sterile  gauze  held  in  place  by  ad- 
hesive straps.     All  iodin  should  be  washed  off  with  alcohol  to  avoid  irri- 
tation or  an  actual  dermatitis. 

Syringe  Method. — 1.  The  technic  is  practically  the  same  as  that 
just  described,  except  that  the  injection  is  given  with  a  syringe. 


METHODS    OF    INOCULATION 


753 


Manufacturing  concerns  market  their  products  in  syringes  all  ready 
for  injection.  When  injecting  tetanus  antitoxin,  it  is  necessary  to 
empty  the  syringe  into  another  sterile  syringe,  as  shown  in  the  accompany- 
ing illustration  (Fig.  143),  which  will  fit  an  appropriate  needle.  Since 
the  plunger  of  the  purchased  syringe  oftentimes  adheres  to  the  barrel 
and  renders  the  injection  jerky  and  difficult,  I  frequently  transfer  the 
serum  to  a  sterile,  all-glass  syringe  which  I  know  will  work  smoothly  and 
satisfactorily. 


FIG.  143. — INTRASPINAL  INJECTION  BY  MEANS  OF  A  SYRINGE. 
The  line  indicates  the  crest  of  the  ilium,  and  usually  passes  between  the  third 
and  fourth  lumbar  vertebrae,  which  is  the  proper  point  for  inserting  the  needle.  The 
site  of  injection  has  been  painted  with  tincture  of  iodin  after  cleansing  with  soap, 
hot  water,  and  alcohol.  An  assistant  holds  the  patient  to  prevent  sudden  jerking 
and  possible  accident. 

2.  Lumbar  puncture  is  performed  as  for  the  gravity  method  while 
blood-pressure  observations  are  being  made.  When  sufficient  fluid  has 
been  removed,  the  loaded  syringe  is  attached  to  the  needle  and  the  in- 
jection slowly  given.  The  physician  is  frequently  tempted  to  inject 
the  serum  and  complete  the  operation  quickly,  but  it  is  better  to  inject 
it  in  amounts  of  0.5  to  1  c.c.  every  half  to  one  minute,  being  guided  by 
the  blood-pressure  readings  and  general  condition  of  the  patient.  If 
the  pressure  falls  below  20  mm.  of  mercury  or  other  symptoms  of  col- 
48 


754  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

lapse  appear,  the  fluid  may  be  drained  from  the  canal  by  gentle  suction 
with  the  piston.  This  is  usually  impossible  when  the  manufacturers' 
syringe  is  used.  Otherwise  the  syringe  is  detached  and  the  fluid  col- 
lected in  tubes  until  the  patient's  condition  improves  and  the  injection 
is  resumed  or  the  needle  removed. 

At  times  the  patient  is  so  restless  that  this  slow  method  is  not  feas- 
ible. In  such  instances  the  physician  should  make  the  injection  as 
slowly  as  possible,  endeavoring  to  put  into  the  canal  as  much  serum  as 
fluid  was  removed  or  at  least  a  reasonable  amount. 


ANTITOXIC  IMMUNIZATION 

SERUM  TREATMENT  OF  DIPHTHERIA 

The  discovery  of  diphtheria  antitoxin  and  its  use  in  the  treatment 
of  this  infection  constitute  one  of  the  triumphs  of  modern  medicine. 

Twenty  years  ago  diphtheria  was  one  of  the  most  dreaded  of  diseases, 
accompanied  ordinarily  by  a  mortality  of  at  least  30  per  cent.,  while  the 
loss  of  life  from  the  laryngeal  form  of  the  disease,  particularly  after 
tracheotomy,  was  simply  appalling. 

Shortly  after  Roux  and  Yersin  (1888)  had  demonstrated  that  the 
symptoms  of  diphtheria  were  due  largely  to  a  soluble  poison  or  toxin  se- 
creted by  the  bacilli,  Ferran,  and  later  Fraenkel  and  Brieger  (1890), 
undertook  experiments  in  active  immunization  against  diphtheria. 
About  the  same  time  von  Behring  discovered  the  antitoxin,  and  in  a 
series  of  extensive  researches  with  Wernicke  he  established  experiment- 
ally its  prophylactic  and  therapeutic  value  in  diphtheria.  The  first 
attempt  to  apply  this  discovery  to  the  cure  of  this  infection  of  the  human 
being  was  made  in  von  Bergmann's  clinic  (1891).  The  results,  while 
encouraging,  were  not  altogether  satisfactory,  owing  largely  to  the  fact 
that  the  serums  were  weak  and  the  doses  given  too  small.  The  dis- 
covery, however,  resulted  in  creating  an  extraordinary  stimulus  to  re- 
searches in  immunity,  and  during  the  following  two  years  more  powerful 
serums  were  prepared,  so  that  in  1896  a  marked  drop  in  the  mortality  of 
diphtheria  was  apparent  in  those  places  where  the  antitoxin  was  being  used. 

Since  then  diphtheria  antitoxin  has  been  the  means  of  saving  count- 
less thousands  of  lives,  and  the  treatment  of  diphtheria,  instead  of  being 
a  reproach  to  medicine,  has  become  the  model  of  what  the  scientific 
treatment  of  an  infectious  disease  ought  to  be.  Statistics  and  the  in- 
dividual experiences  of  those  especially  engaged  in  the  treatment  of 
diphtheria  show  that  when  the  antitoxin  is  used  on  the  first  day  of  the 


SERUM    TREATMENT   OF   DIPHTHERIA  755 

disease,  practically  no  mortality  occurs.  Parents  and  guardians  should 
be  taught  this  fact,  and  cautioned  to  seek  medical  advice  promptly  when- 
ever a  child  complains  of  sore  throat.  While  the  use  of  diphtheria  anti- 
toxin is  still  decried  and  opposed  by  a  few  members  of  the  medical  pro- 
fession,— and  this  is  not  to  be  wondered  at  when  it  is  remembered  that 
cowpox  vaccination  still  has  its  opponents, — it  is  at  least  to  be  hoped 
that  no  physician  will  deprive  a  patient  suffering  from  diphtheria  of  the 
benefits  to  be  derived  from  the  antitoxin  treatment.  In  the  absence  of 
special  contraindications  the  refusal  or  neglect  to  use  antitoxin  in  the  treat- 
ment of  diphtheria  would,  in  the  opinion  of  most  physicians,  constitute  an 
act  of  criminal  negligence  and  malpractice. 

Preparation  of  Diphtheria  Antitoxin. — The  methods  of  preparing  and  stand- 
ardizing diphtheria  antitoxin  are  given  in  Chapter  XIV.  Briefly,  these  con- 
sist in  the  preparation  of  a  strong  toxin  by  cultivating  a  virulent  strain  of  the  bacillus 
in  a  suitable  broth  for  ten  days  or  two  weeks;  the  culture  is  then  passed  through  a 
porcelain  filter,  which  retains  the  bacilli,  the  filtrate  being  a  strong  solution  of  toxin. 
This  is  standardized  by  the  physiologic  test  of  determining  its  action  upon  guinea- 
pigs,  the  minimal  lethal  dose  (M.  L.  D.)  or  toxin  unit  being  the  amount  of  toxin  that 
will  cause  the  death  of  a  250-gram  guinea-pig  in  four  days. 

Strong  and  healthy  horses  that  have  been  proved  by  the  tuberculin  and  mallein 
tests  to  be  free  from  tubercle  or  glanders,  are  then  continuously  immunized  by  a 
series  of  injections  of  the  toxin.  The  first  doses  are  guarded  by  the  simultaneous 
injection  of  antitoxin,  but  after  from  four  to  six  months  the  animals  are  able  to  tolerate 
enormous  quantities  of  pure  toxin.  The  horses  are  then  bled  aseptically  from  a  jugu- 
lar vein,  and  the  separated  blood-serum,  preserved  with  a  small  percentage  of  an 
antiseptic,  becomes  the  antitoxin  of  commerce. 

Many  manufacturing  concerns  market  a  concentrated  diphtheria  antitoxin 
prepared  by  precipitating  the  globulin  fraction  of  the  raw  serum  with  ammonium 
sulphate,  and  redissolving  it  in  a  minimal  quantity  of  salt  solution.  The  globulins 
carry  with  them  most  of  the  antitoxin,  and  in  this  manner  a  serum  may  be  concen- 
trated so  that  a  large  number  of  units  are  contained  in  a  small  bulk  of  fluid,  obviously 
a  most  desirable  feature  in  the  treatment  of  diphtheria.  Further  than  this,  it  has  been 
observed  that  these  concentrated  antitoxins  are  much  less  likely  to  produce  serum 
sickness,  a  train  of  symptoms  due  to  substances  present  alike  in  normal  and  in 
antitoxic  horse  serum,  and  independent  of  the  antitoxin. 

The  antitoxic  unit  is  the  smallest  amount  of  serum  that  will  just  neutralize  100 
times  the  minimal  lethal  dose  of  toxin  for  a  250-gram  guinea-pig.  The  adoption  of 
such  a  standard  enables  us  to  attain  some  accuracy  in  dosage.  Before  its  introduction 
antitoxin  was  given  in  so  many  cubic  centimeters,  just  as  antimeningococcus  and 
antistreptococcus  serums  are  given  today,  but  since  some  horses  produce  more 
potent  serums  than  others,  the  results  were  quite  irregular.  At  the  present  time  an 
antitoxic  serum  is  marketed  according  to  the  number  of  units  it  contains,  irrespective 
of  the  actual  amount  of  serum,  the  constant  endeavor  being  to  produce  as  potent  a 
product  as  possible. 

Since  antitoxin  gradually  deteriorates  with  time,  physicians  should  carefully 
observe  the  date  printed  upon  each  package  of  antitoxin,  which  is  the  time  limit 
calculated  by  the  manufacturers  beyond  which  they  do  not  guarantee  that  the  full 
antitoxic  strength  is  maintained. 


756  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

Nature  of  Diphtheria. — In  the  great  majority  of  cases  diphtheria  is 
a  local  infection  of  some  portion  of  the  upper  respiratory  tract.  The 
bacilli  are  usually  inhaled,  find  lodgment  upon  a  mucous  membrane, 
and  secrete  a  toxin  that  produces  necrosis  of  the  cells  of  the  mucosa  and 
effectually  resists  phagocytosis  of  the  bacilli.  From  this  area  of  infec- 
tion, which  now  becomes  a  prolific  source  of  toxin  production,  the  toxin 
or  poison  is  absorbed  by  the  body-fluids,  and  the  resulting  toxemia  is 
chiefly  responsible  for  most  of  the  symptoms  of  the  disease. 

Other  microorganisms,  such  as  staphylococci,  pneumococci,  and 
streptococci,  which  may  be  unable  to  infect  a  healthy  mucous  membrane, 
readily  multiply  in  the  necrotic  tissue  and  add  to  the  severity  of  the 
local  lesion,  the  lymphadenitis,  and  the  toxemia. 

Rarely  the  diphtheria  bacilli  gain  access  to  the  blood-stream.  The 
severity  and  danger  of  diphtheria  are  dependent  primarily  upon  the 
strength  and  amount  of  toxin  produced  by  the  bacilli,  and  secondarily 
upon  the  size  and  location  of  the  primary  lesion  and  the  amount  of  anti- 
toxin present  in  the  patient's  blood.  A  lesion  in  the  larynx  is  far  more 
dangerous  than  one  of  equal  size  on  a  tonsil,  because  in  the  former  the 
edema  and  necrotic  exudate  obstruct  the  trachea  and  may  produce 
death  by  suffocation.  On  the  other  hand,  the  size  of  the  local  lesion  alone 
is  not  an  indication  of  the  severity  of  the  infection,  because  virulent 
bacilli  in  a  small  patch  may  produce  more  toxin  than  less  virulent  ones 
in  a  larger  area,  and  the  degree  of  local  tissue  necrosis  is  not  an  absolute 
indication  of  the  toxicity  of  the  soluble  poison.  Other  things  being 
equal,  the  patient  who  has  most  antitoxin,  either  naturally  or  acquired 
as  the  result  of  a  previous  injection  with  antitoxin  or  of  an  attack  of 
diphtheria,  will  present  least  evidences  of  toxemia,  although  the  bacilli 
causing  the  infection  may  be  most  virulent.  For  example,  the  highly 
virulent  strain  of  diphtheria  bacillus  used  extensively  in  the  past  eighteen 
years  in  the  production  of  antitoxin  was  isolated  by  Park  and  Williams 
from  the  throat  of  a  patient  presenting  no  clinical  symptoms  other  than 
redness  of  the  fauces  and  slight  toxemia. 

The  primary  lesion  of  diphtheria  is  usually  located  in  the  throat 
(tonsils,  uvula,  larynx),  and  frequently  in  the  nose;  more  rarely  the 
ears,  conjunctiva,  vulva,  prepuce,  and  wounds  are  the  seats  of  primary 
infection. 

Treatment  of  Diphtheria. — If  we  were  always  certain  of  seeing  our 
patients  on  the  first  day  of  their  illness,  and  if  the  disease  could  always 
be  diagnosed  in  this  stage,  the  treatment  of  diphtheria  would  resolve 
itself  into  an  immediate  dose  of  antitoxin  and  rest  in  bed  for  two  or  three 


SERUM   TREATMENT   OF   DIPHTHERIA  757 

weeks.  But  patients  frequently  come  under  treatment  comparatively 
late  in  the  disease,  or  the  true  nature  of  the  condition  may  not  be  fully 
diagnosed  at  first  and  treatment  thus  be  delayed.  Diphtheria  is,  there- 
fore, still  to  be  regarded  as  a  dangerous  infection,  and  while  the  proper 
use  of  antitoxin  constitutes  the  most  important  part  of  the  treatment, 
local  applications,  general  constitutional  measures,  and  the  management 
of  the  various  conditions  that  may  complicate  the  disease  are  all  to  be 
considered.  Here  we  will  discuss  only  the  serum  treatment  of  the  dis- 
ease. 

Bearing  in  mind  the  pathology  of  diphtheria,  serum  treatment  aims 
to  fulfil  the  following  primary  indications : 

1.  To  neutralize  all  free  toxin  circulating  in  the  body  fluids,  and 
also  to  neutralize,  as  much  as  possible,  the  toxin  that  has  already  united 
with  the  tissue-cells. 

2.  To  cause  the  destruction  or  removal  of  the  bacilli  producing  the 
toxin  as  quickly  as  possible. 

3.  To  furnish  the  patient  with  sufficient  excess  of  antitoxin  to  neu- 
tralize the  toxin  as  rapidly  as  it  is  produced  until  the  virulent  bacilli 
disappear. 

Action  of  Diphtheria  Antitoxin. — Diphtheria  antitoxin  best  fulfils 
these  requirements.  In  fact,  there  are  no  substitutes.  In  former  days 
the  powerful  and  irritant  caustics  and  germicides  that  were  freely  ap- 
plied to  the  throat,  instead  of  limiting  the  disease  and  destroying  the 
bacilli,  probably  actually  encouraged  its  extension  by  excoriating  and 
depressing  the  resistance  of  the  surrounding  mucous  membranes. 

1.  The  chief  action  of  antitoxin  is  just  what  its  name  implies,  namely, 
a  substance  that  neutralizes  the  toxin.  This  is  regarded  as  a  chemical 
reaction  analogous  to  the  neutralization  of  an  acid  by  an  alkali.  When 
the  antitoxin  molecule  has  united  with  the  toxin  molecule,  it  is  believed 
that  the  toxin  is  neutralized  and  that  both  are  rendered  inert.  As  the 
result  of  experimental  studies,  however,  we  know  that  this  union  is  not 
always  a  firm  one,  and  it  is  possible  for  the  toxin  to  become  dissociated 
and  attack  body-cells  or  other  molecules  of  antitoxin,  thus  explaining  in 
part  the  necessity  for  giving  quite  large  doses  of  antitoxin — doses  that 
are  out  of  all  proportion  to  what  we  would  expect  to  be  necessary, 
when  considered  weight  for  weight  between  guinea-pig  and  man,  to 
effect  complete  neutralization  of  the  toxin. 

It  is  reasonable  to  presume,  and  may  be  accepted  as  true,  that  a 
stronger  affinity  exists  between  diphtheria  toxin  and  antitoxin  than  be- 
tween body-cells  and  toxin.  Just  as  the  union  between  this  toxin  and 


758  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

its  antitoxin  is  somewhat  unstable,  so,  in  like  manner,  it  is  probable  that 
the  union  of  toxin  and  body-cells  is  not  so  firm  but  that  it  may,  during 
an  early  stage, -be  dissociated  to  some  extent  by  the  more  attractive 
antitoxin.  This  factor  is  to  be  borne  in  mind  in  the  treatment  of  diph- 
theria, and  is  an  additional  argument  for  the  administration  of  large 
doses  of  the  serum. 

2.  Antitoxin  pure  and  simple  does  not,   however,   constitute  the 
only  factor  of  value  in  antidiphtheric  serum.     While  the  pure  antitoxin 
neutralizes  the  toxin,  it  has  no  injurious  action  on  the  bacilli  themselves, 
and,  indeed,  it  is  said  that  the  bacilli  may  live  and  multiply  in  the  pres- 
ence of  large  amounts  of  antitoxin.     How,  then,  are  we  to  explain  the 
gradual  disappearance  of  the  membranous  exudate  and  the  bacilli  at  the 
local  site  of  infection?     It  has  been  shown  experimentally  that  virulent 
diphtheria  bacilli  resist  phagocytosis.     This  condition  is  probably  due 
to  an  actual  leukotoxic  action  of  the  diphtheria  toxin,  aided  by  an  ag- 
gressin-like  action  of  the  toxin  which  neutralizes  opsonin,  and  in  this 
manner  prevents  phagocytic  activity.     I  in  common  with  other  ob- 
servers, have  shown  that  the  antiserum  as  ordinarily  produced  neutral- 
izes the  antiphagocytic  action  of  diphtheria  bacilli,  and  enables  the 
polynuclear  leukocytes  to  ingest  them  readily.1    Whether  this  is  brought 
about  through  neutralization  of  the  toxin  by  antitoxin,  or  is  due  to  the 
presence  of  an  immune  opsonin  (bacteriotropin)  that  lowers  the  re- 
sistance of  the  bacilli,  or  to  the  presence  of  an  anti-aggressin  that  neu- 
tralizes the  intrinsic  defensive  mechanism  of  the  bacilli  and  thus  favors 
phagocytosis,  I  am  unable  to  state,  but  probably  all  three  factors  are 
operative. 

3.  As  ordinarily  prepared,  diphtheria  antitoxin  possesses  little  or  no 
bacteriolytic  activity.     I  have  found,  however,  that  antitoxin  will  fix 
complement  with  an  antigen  of  diphtheria  bacilli,2  indicating,  therefore, 
the  presence  of  bacteriolytic  amboceptors.     Serums  produced  by  im- 
munizing horses  with  unfiltered  cultures  of  diphtheria  bacilli  instead  of 
with  the  filtered  toxin  alone  have  been  advocated  for  the  general  and 
the  local  treatment,  in  the  hope  of  securing  lysis  of  the  infecting  bacilli. 
Certainly  it  would  appear  wise  to  raise  the  bacteriotropic  and  bacterio- 
lytic content  of  an  immune  serum  by  injecting  the  horses  occasionally 
with  unfiltered  cultures,  for  it  is  highly  probable  that  the  action  of  the 
antiserum  depends  not  only  upon  an  antitoxin,  but  also,  to  some  extent 
at  least,  upon  a  bacteriotropin  and  possibly  a  bacteriolysin. 

Administration  of  Diphtheria  Antitoxin. — Antitoxin  is  usually  ad- 
1  Jour.  Med.  Research,  1912,  xxvi,  373.        2  Jour.  Infect.  Dis.,  1912,  xi,  44. 


SERUM    TREATMENT   OF   DIPHTHERIA  759 

ministered  by  subcutaneous  injection  into  the  tissues  of  the  back,  ab- 
domen, or  buttocks.  Experimental  studies  have  tended  to  show  that 
complete  absorption  does  not  occur  until  forty-eight  hours  after,  al- 
though it  is  common  clinical  experience  to  observe  improvement  take 
place  during  the  first  twenty-four  hours  after  injection. 

As  will  be  emphasized  later,  it  is  highly  desirable  and  necessary  "to  get 
antitoxin  into  the  circulation  as  soon  as  possible  after  infection  has  occurred. 
Usually  this  is  best  accomplished  by  giving  antitoxin  to  every  patient 
even  suspected  of  being  diphtheric,  and  making  the  diagnosis  afterward; 
the  next  best  method  is  to  administer  the  antitoxin  in  such  manner  as  to 
favor  quick  absorption.  For  this  reason  intramuscular  and  intravenous 
injection  should  be  resorted  to  in  all  severe  cases.  As  pointed  out  in 
Chapter  XXXI,  the  Schick  reaction  in  diphtheria  has  indicated  that 
diphtheria  toxin  may  be  dissociated  from  tissue-cells  by  large  doses  of 
antitoxin.  Park  and  his  associates  have  shown  experimentally  by  this 
reaction  that  20,000  units  of  antitoxin  given  subcutaneously  were  neces- 
sary to  yield  an  effect  equal  to  1000  units  given  intravenously. 

Intramuscular  injections  into  the  muscles  of  the  buttocks  are  just  as 
readily  given  as  subcutaneous  injections,  and  are  probably  no  more  pain- 
ful to  the  average  patient.  It  insures  quicker  absorption,  and  may, 
indeed,  be  adopted  as  a  routine  practice. 

Intravenous  injections  are  far  more  difficult,  especially  in  children 
with  fat  arms  and  feeble  circulations.  An  anesthetic  or  ten  minutes1 
struggling  may  do  the  patient  harm,  and  unless  the  injection  can  be 
given  with  little  disturbance  and  danger,  the  serum  should  be  given  by 
intramuscular  injection.  Not  infrequently,  however,  an  intravenous 
injection  yields  splendid  results  in  severe  and  apparently  hopeless  cases, 
and  in  older  children  and  adults  this  route  of  administration  should  be 
considered. 

The  syringe  method  is  well  adapted  for  the  intravenous  injection  of 
antitoxin,  as  the  bulk  method  of  serum  is  usually  small,  especially  if  a 
concentrated  antitoxin  is  being  used. 

The  technic  of  these  injections  has  previously  been  described. 

Antitoxin  has  also  been  given  by  the  mouth  and  even  by  rectal  in- 
jection. The  presence  of  a  preservative,  usually  phenol,  renders  the 
oral  administration  objectionable.  While  a  therapeutic  effect  may  be 
secured  after  large  doses  have  been  swallowed,  there  are  very  few  oc- 
casions when  this  should  be  the  method  of  choice. 

Importance  of  Early  Treatment. — The  most  important  point  to  be  ob- 
served in  the  treatment  of  diphtheria  is  to  give  antitoxin  at  once.  It  may 


760  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

be  fatal  to  wait  for  the  result  of  a  culture,  except  perhaps  in  the  case  of  the 
mildest  of  infections.  In  fact,  the  necessity  for  early  administration  of 
antitoxin  cannot  be  overestimated.  When  once  suspicion  is  aroused,  anti- 
toxin should  be  given  at  once  and  the  diagnosis  may  follow.  A  few  hours 
may  make  an  enormous  difference  in  the  prognosis  of  any  case,  and  while 
a  dose  of  2000  units  of  antitoxin  may  prove  of  the  utmost  value  in 
checking  diphtheria,  it  will  do  no  harm  whatever  in  case  the  disease  is 
not  present. 

It  is  true  that  a  physician  will  naturally  hesitate  to  administer  anti- 
toxin unnecessarily;  nevertheless,  diphtheria  is  frequently  a  difficult 
disease  to  diagnose  clinically,  and  is  quite  likely  to  be  mistaken  for  tonsil- 
litis. For  this  reason  many  physicians  prefer  to  wait  for  the  result  of  a 
culture,  and  this  is  proper,  provided  that  the  patient,  especially  in  the 
case  of  a  child,  is  given  the  benefit  of  the  doubt  by  receiving  2000  units 
of  antitoxin.  It  is  to  be  emphasized  that  a  single  negative  culture  does  not 
exclude  diphtheria.  As  ordinarily  made,  about  20  per  cent,  of  primary 
cultures  from  genuine  cases  of  diphtheria  fail  to  show  the  presence  of 
bacilli,  whereas  subsequent  cultures  will  show  them  to  be  present  in 
large  numbers.  A  primary  negative  culture  is  most  likely  to  be  obtained 
from  a  patient  having  a  heavy  exudate,  as  the  physician  may  rub  lightly 
over  the  membrane,  culturing  the  microorganisms  of  secondary  infection 
and  overlooking  the  diphtheria  bacilli  in  the  depths  of  the  membrane 
adjacent  to  the  diseased  mucous  membrane.  To  wait  another  twenty- 
four  hours  for  a  second  culture  still  further  reduces  the  patient's  chances 
for  recovery.  In  the  vast  majority  of  instances,  therefore,  antitoxin  should 
be  given  at  once  and  repeated  as  often  as  is  necessary  until  the  correct  diag- 
nosis is  established,  and  not  one  but  at  least  two  successive  negative  cultures 
should  be  obtained  before  diphtheria  is  to  be  excluded  with  any  reasonable 
degree  of  safety. 

The  following  table,  compiled  from  the  annual  reports  of  the  Phila- 
delphia Hospital  for  Contagious  Diseases,  shows  the  decided  influence 
of  early  treatment  upon  the  mortality  of  diphtheria.  This  table  com- 
prises cases  of  diphtheria  alone,  and  does  not  include  cases  complicated 
by  other  diseases,  such  as  scarlet  fever  and  measles.  It  is  worthy  of 
special  notice  that  of  741  cases  treated  with  antitoxin  on  the  first  day  of  the 
disease,  not  one  died.  Ker,1  in  an  exceptionally  rich  experience,  has 
never  seen  a  fatal  result  occur  in  a  case  that  developed  in  a  hospital  and 
in  which  antitoxin  was  administered  on  the  first  day  of  the  disease. 
1  Infectious  Diseases,  1909,  Oxford  Med.  Press. 


SERUM    TREATMENT    OF    DIPHTHERIA 


761 


TABLE  28.— MORTALITY  OF  DIPHTHERIA  ACCORDING  TO  THE  DAY 

OF  ADMISSION  (WHICH  ALSO  INCLUDES  THE  TIME  OF  GIVING 

THE  ANTITOXIN)  IN  THE  PHILADELPHIA  HOSPITAL 

FOR  CONTAGIOUS  DISEASES 


YEAB 

TOTAL 

NUMBER 
OF  CASES 

MORTALITY  ACCORDING  TO  THE  DAY  ON  WHICH  ANTITOXIN 
WAS  FIRST  INJECTED 

AVERAGE 
MORTALITY 
IN  DIPH- 
THERIA 
ALONE 

First 
Day 

Second 
Day 

Third 
Day 

Fourth 
Day 

Fifth 
Day 

Sixth 
Day 

After 
Sixth 
Day 

1904.. 
1905  

712 
862 
1239 
1426 
2153 
1870 
1895 
1676 
1273 

0 

0 

0 
0 
0 

•  0 

0 
0 

3.92 

4.09 

4.43 
3.45 
6.62 
4.61 
5.50 
6.91 
2.92 
6.51 

13.72 
9.22 
12.90 
4.7 
7.13 
5.13 
9.41 
6.33 
6.52 

17.54 
16.66 
10.85 
10.60 
10.72 
8.41 
7.12 
8.0 
6.87 

14.75 

13.04 
13.08 
11.71 
9.35 
13.74 
11.04 
9.53 
4.76 

19.44 

9.52 
29.41 
21.43 
7.25 
6.04 
7.22 
14.97 
12.5 

12.94 

22.89 
6.09 
23.96 
13.33 
8.33 
2.66 
9.63 
18.8 

10.81 
10.09 
8.70 
8.55 
6.60 
6.42 
6.86 
6.02 
7.63 

1903-07... 
1908  
1909   . 

1910  
1911  
1912  
1913  

Average.  .  . 

13106 

0.4 

5.0 

8.3 

10.7 

11.2 

14.2 

13.1 

7.96 

It  is  generally  believed  that  the  paralyses  of  diphtheria  are  due  to  a 
toxone,  and  not  to  the  true  toxin.  Ehrlich  believes  toxone  to  represent 
a  late  secretory  product  of  the  diphtheria  bacillus,  whereas  others  regard 
it  as  a  modified  toxin.  It  is  certainly  apparent,  however,  that  the  bacilli 
should  be  gotten  rid  of  as  soon  as  possible,  so  as  to  eliminate  the  possi- 
bility of  toxone  production.  This  is  best  accomplished  by  the  early  use 
of  large  doses  of  antitoxin,  aided  possibly  by  judicious  local  treatment. 

Dosage  of  Diphtheria  Antitoxin. — While  it  is  now  generally  agreed 
that  the  doses  of  from  100  to  200  units,  such  as  were  commonly  given 
during  the  early  years  of  antitoxin  therapy,  were  far  too  small,  there  is 
still  some  difference  of  opinion  regarding  the  proper  doses  to  employ. 
Since  the  severity  of  the  disease  varies  so  markedly,  no  hard  and  fast 
rules  can  be  given.  The  physician  who  has  a  clear  idea  of  the  nature 
of  diphtheria  and  of  the  action  of  antitoxin,  and  knows  what  to  expect 
of  the  latter  in  the  treatment  of  the  disease,  should  have  no  difficulty 
in  properly  treating  a  case  of  diphtheria. 

It  is  to  be  emphasized,  however,  that  while  antitoxin  constitutes  the 
most  important  part  of  the  treatment  of  diphtheria,  it  is  not  usually  the 
whole  treatment.  Absolute  rest  in  bed,  a  generous  diet,  combined  with 
the  use  of  tonics  and  local  applications,  are  all  part  of  the  treatment. 
Special  treatment  of  the  laryngeal  form  of  the  disease  and  the  treatment 
of  complications  are  matters  of  considerable  importance  that  influence 
the  prognosis  in  a  given  case.  I  may  mention,  in  passing,  the  value  of 


762  PASSIVE    IMMUNIZATION— SERUM    THERAPY 

continuous  enteroclysis  of  normal  salt  solution  during  the  early  periods 
of  severe  infections;  this  appears  to  dilute  the  toxins  and  aid  in  their 
destruction  and  excretion. 

In  administering  antitoxin  the  physician  must  be  guided  by  the 
clinical  condition  of  the  patient,  as  we  have  as  yet  no  practical  labora- 
tory method  for  estimating  the  degree  of  the  toxemia.  Treatment  may 
be  regarded  as  satisfactory  when — 

1.  The  local  patch  of  exudate  has  ceased  to  spread  and  shows  indica- 
tions of  disappearing. 

2.  The  general  condition  of  the  patient  is  improved,  i.e.,  the  toxemia 
is  decreased,  the  pulse  grows  stronger  and  more  regular,  and  the  patient 
feels  more  comfortable. 

The  temperature  is  not  a  reliable  guide,  for  not  infrequently  in 
severe  infections  it  may  be  normal  or  subnormal  throughout. 

So  long  as  the  exudate  shows  no  signs  of  loosening  and  disappearing, 
but  tends  to  spread,  and  so  long  as  the  general  condition  remains  unim- 
proved or  grows  worse,  large  amounts  of  antitoxin  should  be  given. 
No  case  should  be  regarded  as  hopeless  until  death  supervenes. 

Every  case  of  diphtheria  is  to  be  treated  individually,  rather  than  by 
any  set  rule.  The  amount  of  antitoxin  given  in  the  initial  dose,  and  in 
subsequent  doses  as  well,  is  dependent  on  the  following  factors : 

1.  The  situation  and  extent  of  the  lesion. 

2.  The  general  condition  of  the  patient. 

3.  The  day  of  the  disease. 

4.  The  age  of  the  patient. 

1.  The  Situation  and  Extent  of  the  Lesion. — In  ordinary  tonsillar 
diphtheria  in  which  there  is  a  small  patch  on  one  tonsil  and  which  is  first 
seen  on  the  second  day  of  the  disease,  the  initial  dose  should  be  at  least 
5000  units.  When  the  exudate  involves  the  pillars  of  the  fauces,  the 
uvula,  the  posterior  pharyngeal  wall,  or  is  well  forward  on  the  palate, 
this  dose  should  be  doubled,  and  10,000  units  be  given. 

Cases  that  present  laryngeal  symptoms  in  addition  to  faucial  lesions 
should  never  receive  less  than  12,000  units.  In  well-marked  laryngeal 
diphtheria  with  dyspnea  and  partial  suffocation  at  least  20,000  units 
should  be  given,  preferably  by  intramuscular  or  intravenous  injection. 

In  nasal  diphtheria  a  distinction  must  be  made  between  those  cases 
that  exhibit  merely  a  dirty,  chronic  discharge  containing  bacilli,  in  which 
2000  units  may  suffice,  and  those  that  present  an  actual  membrane  ac- 
companied by  well-marked  toxic  symptoms,  when  a  large  amount  of 
serum — at  least  10,000  units — should  be  given.  Owing  to  the  ready 


SERUM   TREATMENT   OF   DIPHTHERIA  763 

absorption  of  toxin  by  the  nasal  mucosa  a  small  patch  in  the  nose  may  be 
accompanied  by  severe  general  toxemia  and  frequently  requires  ener- 
getic treatment. 

In  diphtheria  of  the  eye,  vulva,  or  of  wounds  relatively  large  doses 
should  be  given— at  least  from  10,000  to  20,000  units. 

All  these  doses  must  be  regarded  merely  as  suggestions  for  the  initial 
dose  in  cases  seen  on  about  the  second  day  of  the  disease.  Physicians 
with  extensive  hospital  experience,  for  example,  Woody  and  McCullom, 
generally  favor  large  doses,  and  while  in  private  practice  the  question 
of  expense  and  economy  may  be  a  factor,  the  physician  will  be  wise  to 
err  on  the  side  of  safety  and  give  a  little  too  much  serum  rather  than  too 
little,  especially  in  the  first  dose,  when  so  much  depends  upon  how  soon 
antitoxin  is  introduced  into  the  body-fluids. 

2.  The  general  condition  of  the  patient,  or  the  effect  which  the  tox- 
emia will  have  on  the  patient,  is  highly  important  in  estimating  the 
dosage  of  antitoxin.     A  patient  who  is  pale,  drowsy,  prostrated,  and 
has  a  weak  and  irregular  pulse;   who  has  large  masses  of  glands  around 
the  neck,  or  who  has  marked  albuminuria,  will  require  a  much  larger 
dose  than  one  who  presents  none  of  these  signs.     Two  persons  of  about 
the  same  age  and  suffering  from  the  same  lesions  may  show  very  differ- 
ent degrees  of  toxemia.     In  the  severe  cases  we  must  administer  the 
maximum  dose  and  repeat  it  at  suitable  intervals  until  an  effect  is  pro- 
duced. 

3.  The  day  of  illness  on  which  the  patient  comes  under  observation 
is  important  in  deciding  the  initial  dose  of  antitoxin.     For  corresponding 
tonsillar  lesions  a  dose  of  2000  units  on  the  first  day  may  do  more  good 
than  5000  units  given  on  the  fourth  day.     Ker  gives  second-day  cases 
of  purely  tonsillar  diphtheria  3000  units,  and  adds  an  additional  1000  on 
the  following  day. 

4.  If  the  age  of  the  patient  exerts  any  influence  at  all  on  dosage,  it  in- 
dicates that  more  antitoxin  should  be 'given  to  children  than  to  adults 
with  corresponding  lesions,  as  the  disease  is  more  fatal  in  children.     So 
far  as  infants  are  concerned,  Ker  seldom  gives  more  than  4000  units  at  a 
single  dose,  which  should  be  an  adequate  amount  when  we  consider  the 
small  size  of  the  patient.     Children  over  one  year  of  age  may  be  given 
from  5000  to  10,000  or  more  units,  depending  upon  the  location  of  the 
lesion  and  the  degree  of  toxemia. 

Repeating  the  Dose. — Whether  or  not  subsequent  doses  of  antitoxin 
will  be  required  is  dependent  upon  the  circumstances  of  the  individual 
case.  In  ordinary  cases  if  on  the  day  after  treatment  is  commenced 


764  PASSIVE   IMMUNIZATION — SERUM    THERAPY 

there  is  no  diminution  in  the  amount  of  membrane  visible  and  the  gen- 
eral symptoms  have  not  improved,  the  dose  should  be  repeated.  If  the 
membrane  has  spread  and  the  toxemia  is  worse,  the  second  dose  should 
be  larger  than  the  first.  In  septic  cases  the  second  dose  may  be  given 
in  from  six  to  ten  hours  after  the  first.  If  the  symptoms  are  less  urgent, 
the  interval  may  be  extended  to  twelve,  but  should  never  exceed  twenty- 
four  hours. 

As  to  the  total  amount  of  serum  to  be  administered,  continued  injec- 
tions at  relatively  short  intervals  are  required  until  improvement  has 
taken  place.  So  long  as  membrane  is  present  and  the  patient  is  toxic 
it  is  probably  worth  while  to  push  the  treatment  unless  these  show  a  ten- 
dency to  clear  away.  Time  must  be  allowed  for  absorption  to  take  place, 
and  the  serum  should  not  be  pushed  so  far  as  to  be  wasted,  and,  quite 
possibly,  excreted  unchanged.  The  remedy  is  expensive,  especially  in 
private  practice,  and  it  is  obviously  desirable  to  have  due  regard  for 
economy.  While,  as  previously  mentioned,  physicians  of  such  wide  ex- 
perience as  McCullom,  of  Boston,  and  Woody,  of  Philadelphia,  fre- 
quently give  200,000  or  more  units  in  severe  cases  of  diphtheria,  others, 
e.  g.,  Ker,  of  Edinburgh,  have  never  given  more  than  64,000  units  to  a 
single  patient,  and,  indeed,  several  of  my  colleagues  of  wide  experience 
claim  that  they  have  secured  excellent  results  in  severe  infections  with 
doses  that  seldom  exceeded  10,000  units. 

Treatment  of  Relapses. — Occasionally,  after  a  patient  has  recovered 
from  an  attack  of  diphtheria  the  infection  recurs  after  several  weeks. 
It  is  in  such  cases  that  the  physician  hesitates  to  administer  antitoxin, 
on  account  of  the  discomforts  occasioned  by  serum  sickness.  It  is  true 
that  serum  sickness  is  more  likely  to  follow  in  these  than  in  primary 
cases,  and  the  very  profuse  and  itchy  eruption,  joint  pains,  and  fever 
do  indeed  render  the  patient  quite  uncomfortable.  Since  a  relapse  is 
usually,  although  not  always,  comparatively  mild,  the  serum  may  be 
dispensed  with  if  there  is  no  involvement  of  the  larynx  and  if  there  is 
not  much  evidence  of  toxemia;  otherwise  full  doses  of  antitoxin  should 
be  given  without  hesitation.  The  subcutaneous  injection  of  0.5  c.c. 
of  the  serum  two  or  three  hours  before  the  main  dose  is  given  may  pos- 
sibly produce  a  condition  of  anti-anaphylaxis  and  ward  off  the  more 
dangerous  symptoms.  If  respiratory  difficulties  should  follow,  a  re- 
injection  of  serum,  atropin,  and  caff ein  should  be  administered  hypo- 
dermically. 

Antitoxin  Sequelae. — A  certain  percentage  of  cases  will  present  a 
group  of  symptoms  that  constitute  the  condition  known  as  serum  sick- 


SERUM    TREATMENT   OF   DIPHTHERIA  765 

ness,  occurring  at  varying  times  following  the  administration  of  anti- 
toxin. This  condition  has  been  shown  to  be  due  to  certain  constituents 
of  horse  serum  other  than  the  antitoxic  antibodies.  It  is  noteworthy 
that  the  serum  of  one  horse  may  cause  more  serum  sickness  than  that  of 
another;  in  general,  concentrated  antitoxins  produce  fewer  cases  than 
do  raw  serum. 

This  condition  is  characterized  by  the  development  of  a  rash  (urti- 
carial,  multiform,  or  scarlatiniform),  mild  fever,  joint  pains,  and  possi- 
bly adenitis.  The  scarlatiniform  rash  may  be  extremely  difficult  to 
differentiate  from  that  of  true  scarlatina,  especially  in  the  wards  of  a 
diphtheria  hospital,  where  outbreaks  of  scarlet  fever  are  not  uncommon. 

This  subject  has  been  considered  in  greater  detail  in  Chapter 
XXVIII  on  Anaphylaxis.  It  may  be  stated  here  that  while  the  patient 
is  decidedly  uncomfortable,  and  even  quite  sick,  for  several  days,  serum 
sickness  is  not  a  dangerous  condition;  the  treatment  is  largely  symp- 
tomatic and  palliative. 

Value  of  Diphtheria  Antitoxin. — At  the  present  day  it  seems  hardly 
necessary  to  introduce  elaborate  statistics  to  prove  the  value  of  anti- 
toxin in  the  prophylaxis  and  treatment  of  diphtheria. 

1.  It  is  generally  admitted  that  most  of  the  reduction  in  the  mortality 
of  diphtheria  cannot  be  attributed  solely  to  the  use  of  antitoxin,  for 
unquestionably  bacteriologic  diagnosis  has  permitted  the  inclusion,  in 
our  statistics,  of  a  certain  number  of  cases  that,  twenty  years  ago,  would 
not  have  been  classed  as  diphtheria.  Generally  speaking,  however,  the 
mortality  of  diphtheria,  considering  all  types  of  infection  coming  under 
observation  at  varying  intervals  after  the  disease  has  developed,  is  at 
least  five  times  less  under  antitoxin  treatment  than  when  it  is  treated  without 
antitoxin.  This  proportion  is  true,  whether  we  compare  the  general 
mortality  before  1896  with  the  present  rate,  or  whether  we  take  a  large 
city  and  compare  the  mortality  under  both  forms  of  treatment  for  a 
single  or  for  several  years.  For  example,  in  Philadelphia  the  mortality 
rates  per  100,000  of  population  for  the  five  years  preceding  the  use  of 
antitoxin  were  as  follows: 

1891 127.4 

1892 : 156.3 

1893 103.9 

1894 122.5 

1895 : • 115.9 

In  five  years  following  the  general  use  of  antitoxin  the  mortality 
rates  per  100,000  population  were  as  follows: 


766  PASSIVE    IMMUNIZATION SERUM    THERAPY 

1906 37.78 

1907 34.60 

1908 33.35 

1909 33.6 

1910..  -.31.7 


In  Philadelphia,  during  the  years  1909-10  and  1911,  the  average  mor- 
tality of  diphtheria  treated  with  antitoxin  in  the  Philadelphia  Hospital 
for  Contagious  Diseases  was  9.9  per  cent.,  and  in  the  private  practice 
of  physicians,  13.07  per  cent.  In  contrast  to  these  figures  is  the  mor- 
tality of  40.34  per  cent,  in  the  private  practice  of  those  physicians 
(fortunately  few)  who  refused  to  give  antitoxin  or  in  those  families  op- 
posed to  its  use. 

From  Table  28  it  will  be  seen  that  the  average  mortality  in  13,106 
cases  of  pure  diphtheria  treated  with  antitoxin  in  the  Philadelphia  Hos- 
pital for  Contagious  Diseases  during  the  past  ten  years  was  only  7.96 
per  cent.  As  stated  elsewhere,  when  this  is  compared  with  the  average 
mortality  of  about  41  per  cent,  when  no  antitoxin  was  used,  it  is  not  diffi- 
cult to  appreciate  the  therapeutic  value  of  the  remedy. 

2.  As  was  previously  stated,  it  is  also  worthy  of  note  that  there  is 
practically  no  mortality  in  diphtheria  cases  receiving  antitoxin  on  the 
first  day  of  illness.     During  nine  consecutive  years  (1904-1913),  covering 
the  treatment  of  741  such  cases  in  the  Philadelphia  Hospital  for  Contagious 
Diseases,  not  a  single  death  occurred.      During  1913  two  of  the  51  first- 
day  cases  died. 

It  will  also  be  noted  from  Table  28  that  the  patient's  chance  for  re- 
covery grows  steadily  less  with  each  day  that  the  administration  of 
serum  is  delayed,  and  this  should  be  evidence  enough  to  convince  any 
right-minded  person  that  we  possess  in  antitoxin  a  remarkable  remedy 
for  the  treatment  of  diphtheria. 

3.  The  influence  of  antitoxin  is  also  noted  in  the  mortality  of  laryn- 
geal  diphtheria.     While  the  mortality  in  this  condition  is  still  high,  ow- 
ing to  the  frequency  and  dangers  of  bronchopneumonia  and  the  necessity 
for  operative  measures,  it  has  been  reduced  at  least  one-half  since  anti- 
toxin came  into  general  use.     Prior  to  1896  the  mortality  was  at  least 
70  per  cent.;   since  then  it  has  been  reduced  to  35  per  cent,  or  less.     As 
shown  in  the  following  table,  of  1207  cases  treated  in  the  Philadelphia 
Hospital  for  Contagious  Diseases,  the  average  mortality  was  35.6  per 
cent. 


SERUM   TREATMENT   OF  DIPHTHERIA 


767 


TABLE  29.— MORTALITY  OF  LARYNGEAL  DIPHTHERIA  (INTUBA- 
TION CASES)  IN  THE  PHILADELPHIA  HOSPITAL  FOR 
CONTAGIOUS  DISEASES 

(This  table  excludes  those  patients  dying  in  the  ambulance  and  moribund 
cases  dying  within  twenty-four  hours.) 


YEAH 

TOTAL 

NUMBER 
OF 

CASES 

MORTALITY  ACCORDING  TO  THE  DAY  UPON  WHICH  INTUBA- 
TION WAS  PERFORMED 

AVERAGE 
MORTALITY 

First 
Day 

Second 
Day 

Third 
Day 

Fourth 
Day 

Fifth 
Day 

Sixth 
Day 

After 
Sixth 
Day 

1903.. 

67 
125 
136 
72 
162 
183 
89 
104 
133 
136 

66.6 

idd.6 

50.0 
30.0 
100.0 

25.3 

39.20 
30.76 
50.0 
29.26 
50.0 
55.0 
76.66 
58.33 
65.91 

21.8 
29.03 
39.39 
55.0 
50.0 
44.18 
33.33 
55.55 
41.93 
63.64 

30.9 
73.68 
50.0 
40.74 
33.33 
38.88 
28.58 
52.63 
48.27 
35.29 

22.5 
33.33 
21.42 
45.45 
30.76 
23.81 
50.0 
36.36 
50.0 
25.0 

70.0* 
16.66 
71.92 
9.09 
53.84 

63.67 

47.82 

45.0 

36.84 
42.30 
28.57 
38.88 
40.0 
36.46 
27.27 
50.0 
68.0 

26.60 
39.20 
36.76 
49.29 
33.95 
42.62 
39.34 
54.80 
48.87 
58.82 

1904 

1905 

1906-07.  .  . 
1908  

1909  

1910  

1911  

1912 

1913 

Average..  . 

1207 

35.6 

Formerly  when  a  child  contracted  diphtheria  the  parents  were 
warned  of  the  likelihood  and  danger  of  the  infection  involving  the  larynx 
and  trachea;  nowadays  this  possibility  is  quite  remote. 

4.  Finally  the  claim  of  the  opponents  of  the  serum  therapy  of  diph- 
theria that  antitoxin  increases  the  percentage  of  paralyses  is  without 
foundation.  While  it  is  true  that  this  percentage  is  somewhat  higher 
than  was  noted  in  former  years,  this  increase  is  to  be  explained  by  the 
fact  that  antitoxin  saves  a  larger  number  of  severe  cases  long  enough 
for  them  to  manifest  paralyses,  and,  second,  by  the  greater  attention 
that  has  recently  been  directed  to  its  milder  forms.  Since  diphtheric 
paralysis  is  regarded  as  caused  by  toxone  or  a  later  secondary  toxic 
product  of  the  bacilli,  the  indications  are  to  rid  the  patient  of  the  bacilli 
as  quickly  as  possible,  and  this  is  best  and  most  surely  accomplished  by 
the  proper  administration  of  antitoxin. 

PROPHYLACTIC  IMMUNIZATION  AGAINST  DIPHTHERIA 
The  subcutaneous  administration  of  relatively  small  doses  of  anti- 
toxin will  usually  confer  a  passive  immunity  against  diphtheria  lasting 
from  two  to  four  weeks. 

The  object  is  to  introduce  antibodies  (antitoxin  and  opsonin)  into  the 
body-fluids  in  order  that  they  may  neutralize  the  toxin  as  rapidly  as  it 
is  produced,  aid  in  the  destruction  of  the  bacilli,  and  thus  protect  the 


768  PASSIVE   IMMUNIZATION — SERUM    THERAPY 

individual  in  case  virulent  bacilli  should  be  inspired  or  otherwise  gain 
access  to  the  tissues. 

The  doses  advised  are  relatively  small,  and  the  injection  does  not 
usually  produce  any  discomfort  other  than  soreness  about  the  site  of  in- 
oculation. For  infants  under  one  year  of  age  500  units  suffice;  for 
older  children  and  adults  from  1000  to  1500  units  should  be  given. 

The  duration  of  this  passive  immunity  is  relatively  short,  owing  to  the 
fact  that  the  antitoxin  is  eliminated  rapidly,  as  it  is  part  and  parcel  of  a 
foreign  serum  that  tends  to  be  excreted  or  destroyed  soon  after  its  intro- 
duction into  the  body.  It  will  endure,  however,  for  at  least  two  weeks, 
and  frequently  longer.  Since  the  incubation  period  of  diphtheria  is 
only  a  matter  of  a  few  days,  this  suffices,  in  the  majority  of  instances,  to 
protect  the  individual. 

The  indications  are  to  immunize  all  persons  who  have  come  in  inti- 
mate contact  with  a  case  of  diphtheria.  If  time  permits  the  Schick  test 
should  be  conducted,  as  only  those  persons  yielding  positive  reactions 
require  the  antitoxin  (see  page  229).  It  is  especially  valuable  in  fam- 
ilies and  small  communities,  such  as  go  to  make  up  hospital  wards 
and  asylums.  The  physician  who  is  attending  a  case  of  diphtheria  in 
a  private  home  should  urge  immunization  upon  all  members  of  the  house- 
hold. 

The  immediate  results  ?,re  usually  good.  The  main  disadvantage  is 
the  short  duration  of  the  immunity,  so  that  no  matter  how  faithfully  it 
is  carried  out,  persons  do  not  remain  immune  for  long  periods  of  time, 
and  accordingly  the  total  morbidity  of  the  disease  is  not  influenced  to 
any  extent.  In  homes  from  which  the  case  of  diphtheria  is  promptly 
removed  to  a  special  contagious  hospital  and  in  which  the  remaining 
members  are  promptly  immunized  the  percentage  of  secondary  cases 
is  practically  nil.  Of  6772  patients  who  were  removed  to  the  Phila- 
delphia Hospital  for  Contagious  Diseases,  the  remaining  members  of 
the  family  not  being  immunized,  secondary  cases  developed  in  164  per- 
sons, or  in  2.4  per  cent.  Of  4063  cases  of  diphtheria  treated  at  home  with 
antitoxin,  the  other  members  of  the  family  not  being  immunized,  sec- 
ondary cases  developed  in  219  persons,  or  in  5.3  per  cent.  Of  639  diph- 
theric patients  treated  at  home  who  did  not  receive  antitoxin  and  where 
immunization  was  not  practised,  secondary  cases  developed  in  151 
persons,  or  23.6  per  cent.  These  figures,  compiled  by  Dr.  A.  A.  Cairns, 
chief  medical  inspector  of  Philadelphia,  and  taken  from  the  annual  re- 
ports of  the  Philadelphia  Bureau  of  Health  for  the  years  of  1909,  1910, 
and  1911,  show  that  the  best  results  are  obtained  when  the  diphtheric 


SERUM   TREATMENT    OF   DIPHTHERIA  769 

patient  is  promptly  removed  to  a  special  hospital  and  the  remaining 
members  of  the  household  are  immunized.  Even  when  the  patient  is 
removed  promptly  there  is  some  danger  of  other  persons  having  been 
infected,  and  immunization  should,  therefore,  always  be  promptly  prac- 
tised. When  the  patient  is  treated  at  home,  other  members  of  the 
household,  even  if  immunized,  are  liable  to  develop  the  infection,  prob- 
ably owing  to  the  fact  that  the  patient  harbors  virulent  bacilli  for  vary- 
ing periods  of  time  after  the  passive  immunity  in  other  persons  has  passed 
away  and  the  quarantine  is  broken.  Certainly  in  those  homes  where 
antitoxin  is  not  used  either  for  therapeutic  or  for  prophylactic  purposes, 
the  percentage  of  secondary  infections  is  so  high  as  to  leave  no  doubt 
as  to  the  value  of  antitoxin. 

In  this  connection  I  may  mention  the  desirability  of  using  an  anti- 
toxin prepared  by  immunization  of  cattle  for  the  general  purpose  of 
prophylaxis,  and  especially  for  the  treatment  of  those  persons  who  are 
hypersensitive  to  horse  serum.  In  these  cases  horse  antitoxin  could  be 
used  later  if  a  person  contracted  diphtheria  without  danger  of  anaphy- 
laxis. 

Behring's  Method  of  Immunization  against  Diphtheria. — Owing  to 
the  fact  that  the  antibodies  produced  through  the  activities  of  our  own 
body-cells  (active  immunization)  persist  for  longer  periods  of  time  than 
those  that  are  introduced  passively  (passive  immunization),  Behring  and 
his  assistants  have  been  working  upon  a  method  of  active  immuniza- 
tion in  diphtheria  whereby  our  own  body-cells  are  to  be  stimulated  to 
produce  our  own  antitoxin  in  sufficient  amounts  to  protect  us  against  the 
disease.  It  has  long  been  known  that  more  or  less  balanced  mixtures 
of  this  kind  produce  immunity  in  animals,  and  in  1907  Theobald  Smith1 
suggested  that  it  might  be  possible  to  employ  this  method  for  the  pur- 
pose of  producing  immunity  in  man.  Subsequently  Smith2  studied  the 
effects  of  injections  of  neutral  mixtures  in  guinea-pigs  and  horses,  and 
again  pointed  out  the  applicability  of  the  method  to  human  beings 

Active  immunization  in  diphtheria  could  probably  be  accomplished 
by  the  administration  of  minute  and  increasing  doses  of  toxin,  but  there 
would  be  some  danger  of  producing  an 'acute  toxemia  or  paralysis,  and 
the  process  may  require  so  much  time  as  to  be  useless  in  the  presence  of 
epidemics. 

Behring's  method,3  accoring  to  his  report  read  before  the  German 

1  Jour.  Med.  Research,  1907,  xvi,  359. 

2  Jour.,  Exper.  Med.,  1909,  xi,  241;  Jour.  Med.  Research,  1-910,  xxiii,  433. 

3  Deutsch.  med.  Wchnschr.,  1913,  xxxix,  873. 

49 


770  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

Convention  on  International  Medicine  in  1913,  is  based  upon  the  prin- 
ciple that  the  union  of  toxin  and  antitoxin  is  not  stable,  and  when  a 
neutral  mixture  of  the  two  is  injected  into  animals,  sufficient  toxin  be- 
comes dissociated  to  unite  with  body  cells  and  stimulate  the  production 
of  antitoxin. 

The  mixture  of  toxin  and  antitoxin  is  known  as  T.-A.,  and  several 
strengths  are  being  used;  of  these,  the  first  dose  possesses  the  lowest 
toxicity  and  is  overneutralized  by  mixing  1  unit  of  antitoxin  with  50 
per  cent,  of  the  L+  dose  of  toxin;  the  second  dose  may  be  about  neutral 
or  65  per  cent,  of  the  L+  toxin  with  1  unit  of  antitoxin;  the  third  dose 
is  usually  slightly  toxic  for  the  guinea-pig  afid  is  prepared  with  80  per 
cent,  of  the  L+  dose  of  toxin  with  1  unit  of  antioxin.  I  generally 
prepare  these  solutions  in  such  proportions  that  the  proper  doses  are 
contained  in  1  c.c.  The  following  is  an  example  of  the  preparation  of 
100  doses  of  each  of  the  three  mixtures  with  a  toxin  having  an  L-f-  dose 
of  0.2  c.c.: 

First  dose:  50  per  cent,  of  the  L+  dose  of  toxin  or  10  c.c.  for  100 
doses. 

Second  dose:  65  per  cent  of  the  L+  dose  of  toxin  or  13  c.c.  for 
100  doses. 

Third  dose:  80  per  cent,  of  the  L-f  dose  of  toxin  or  16  c.c.  for 
100  doses. 

To  each  of  these  is  added  an  amount  of  antitoxin  serum  equal  to 
100  units  and  sufficient  sterile  salt  solution  containing  0.25  per  cent, 
tricresol  to  make  the  total  volume  100  c.c.  After  standing  several  hours 
the  toxicity  of  each  mixture  is  tested  by  subcutaneous  injections  into 
250-gram  guinea-pigs. 

The  dose  of  each  mixture  is  1  c.c.  and  the  injections  are  given  sub- 
cutaneously  at  intervals  of  ten  days. 

The  method  has  now  been  used  for  immunizing  a  large  number  of 
persons,  chiefly  under  the  supervision  of  von  Behring  and  his  assistants.1 
The  natural  antitoxin  content  of  the  blood  is  determined,  usually  by 
the  intracutaneous  method  on  the  guinea-pig,  before  and  after  immuni- 
zation, and  has  shown  uniformly  a  considerable  increase,  which  persists 
over  many  months. 

Local  and  general  reactions  have  been  observed,  especially  in  older 
children  and  adults  with  doses  intended  for  the  new-born;  this  fact  is 
explained  on  the  assumption  of  a  specific  sensitization  in  consequence 

1  See  Semaine  Me"dicale,  1913,  xxxiii,  No.  18;  Berl.  klin.  Wochenschr.,  1914, 
hx,  917;  ibid.,  1914,  lix,  917;  Therap.  Monatssch.,  1913,  xxvii,  No.  11;  Deutsch.  med. 
Wchnschr.,  1913,  xxx,  460;  ibid.,  1913,  xxix,  977;  ibid.,  1913,  xxxix,  1977;  ibid.,  1913, 
xxxix,  2500;  ibid.,  1914,  xl,  13;  ibid.,  1914,  xl,  582. 


SERUM    TREATMENT    OF   DIPHTHERIA  771 

of  the  previous  introduction  of  diphtheria  bacilli  (carriers),  which  latter 
render  the  individuals  hypersensitive  to  the  T.-A.  Reactions  that  are 
regarded  as  non-specific  have  been  observed  in  tuberculous  and  scrofu- 
lous persons,  and  for  the  present  von  Behring  prefers  that  the  use  of  the 
prophylactic  in  such  persons,  as  well  as  in  atrophic  infants  and  infants 
less  than  nine  months  old,  be  regarded  as  contraindicated. 

The  fear  expressed  by  some  that  the  prophylactic  is  contraindicated 
in  those  persons  who  harbor  diphtheria  bacilli  for  fear  of  producing  the 
disease  during  temporary  depression  of  the  defensive  mechanism  has 
been  finally  dissipated  as  the  result  of  practical  experience.  Not  one 
of  the  numerous  bacillus-carriers  that  have  been  injected  with  T.-A. 
have  sickened  with  diphtheria.  Whether  or  not  the  active  immuniza- 
tion with  T.-A.  will  help  them  to  get  rid  of  the  bacilli  is  still  an  open 
question. 

The  subcutaneous  injection  is  recommended  as  the  best  method  of 
administration.  There  can  be  no  doubt  that,  in  many  cases,  a  single 
injection  produces  sufficient  protection.  Such  persons  are,  as  a  rule, 
those  who  have  already  been  sensitized  by  diphtheria  bacilli.  For  the 
ordinary  run  of  cases  at  least  three  injections  should  be  given.  The  first 
injection  then  plays  the  part  of  a  sensitizer.  Experience  shows  that 
sensitization  occurs  after  from  ten  to  fourteen  days,  which  makes  it 
necessary  that  the  second  injection  should  not  be  given  until  after  an 
interval  of  not  less  than  ten  days. 

In  this  country  the  subject  has  been  studied  by  William  H.  Park  and 
his  associates,1  who  found  that  this  form  of  active  immunization  gave 
rise  to  decided  antitoxin  production  in  22  per  cent,  of  susceptible  per- 
sons. The  interval  between  vaccination  and  the  development  of  im- 
munity was  generally  long — as  a  rule,  not  less  than  two  weeks.  Under 
conditions  of  exposure  about  20  per  cent,  of  those  who  failed  to  respond 
were  found  to  develop  clinical  diphtheria.  Only  those  persons  yielding 
positive  Schick  reactions  require  immunization  when  exposed  to  diphtheria 
and  in  immediate  danger.  Antitoxin  alone  may  be  used  or,  if  a  longer 
protection  is  desired  and  time  permits,  the  T.-A.  mixtures  may  be  em- 
ployed. 

The  remedy  has  not  been  used  sufficiently  often  to  enable  us  to  ex- 
press an  opinion  as  to  its  value.  Behring  believes  that  its  proper  use 
may  thoroughly  eradicate  diphtheria.  Obviously,  its  preparation  must 
be  very  carefully  controlled,  and  for  the  present  it  should  be  used  only 
in  institutions  where  thorough  studies  of  the  blood  of  patients  may  be 
made  before  and  after  immunization. 

1  Jour.  Amer.  Med.  Assoc.,  1914,  63,  859;  ibid.,  1915,  65,  2216. 


772  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

SERUM  TREATMENT  OF  TETANUS 

Tetanus  antitoxin  was  discovered  by  Behring  and  Kitasato  in  1892. 
Since  then  it  has  proved  of  great  value  in  the  prevention  of  lockjaw. 
While,  in  general,  authoritative  opinions  regarding  its  curative  value 
vary,  the  statistics  and  the  individual  experience  of  many  investigators 
of  more  recent  years  show  quite  conclusively  that  tetanus  antitoxin  does 
possess  curative  value,  and  is  of  distinct  aid  in  the  treatment  of  tetanus, 
especially  when  the  nature  of  the  disease  is  understood  and  the  serum  is 
administered  accordingly. 

Preparation  and  Standardization  of  Tetanus  Antitoxin. — This  technic  has 
been  described  in  Chapter  XIV.  Briefly,  it  consists  in  immunizing  the  horse 
with  gradually  increasing  doses  of  tetanus  toxin  over  a  period  of  several  months, 
until  the  blood  of  the  animal  contains  the  antitoxin  in  sufficient  quantities  for  thera- 
peutic use.  The  animal  is  then  bled  under  aseptic  precautions,  and  the  serum,  with 
the  addition  of  a  small  amount  of  preservative,  constitutes  the  antitoxin  of  commerce. 
Several  manufacturers  concentrate  the  antitoxin  in  the  same  manner  as  diphtheria 
antitoxin  is  concentrated.  In  view  of  the  very  large  doses  required  in  the  treatment 
of  tetanus  this  is  quite  desirable. 

The  American  unit  is  denned  as  the  amount  of  antitoxin  required  just  to  neu- 
tralize 1000  fatal  doses  of  tetanus  toxin  for  a  350-gram  guinea-pig.  The  United 
States  Government  has  adopted  this  unit,  and  supplies  the  different  producers  with 
standardized  toxin  for.  testing  the  antitoxin. 

Action  of  Tetanus  Toxin. — It  may  be  well  to  recall  briefly  the  main 
features  concerning  the  pathogenesis  of  tetanus,  as  successful  treatment 
depends  upon  a  thorough  understanding  of  these  principles. 

1.  Tetanus  is  a  local  infection  accompanied  and  characterized  by  a 
general  toxemia.     The  bacilli  and  spores  never  gain  access  to  the  blood, 
and  are  never  distributed  through  the  tissues  and  internal  organs,  but 
reside  at  the  local  site  of  infection,  where  they  produce  a  powerful  toxin, 
which,  when  absorbed,  is  responsible  for  the  main  lesions  and  symptoms 
of  the  infection.     Therefore  while  the  blood  of  the  tetanus  patient  is 
sterile,  it  usually  contains  the  toxin.     Neisser  has  produced  tetanus  in 
mice  by  giving  them  subcutaneous  injections  of  the  blood  of  a  tetanus 
patient. 

2.  Tetanus  toxin  has  a  strong  affinity  for  nerve  tissue,  and  this  con- 
stitutes the  most  important  feature  in  the  pathogenesis  of  the  disease. 
The  toxin  is  rapidly  absorbed  from  the  local  site  of  infection  into  the 
blood  and  lymph-streams,  where  it  is  distributed  to  other  muscles  and 
reaches  the  central  nervous  system  indirectly  through  absorption  by 
the  end-plates  of  motor  nerves.     As  expected,  absorption  is  most  likely 
to  occur  along  the  motor  nerves  supplying  the  parts  injured,  and  for  this 
reason  the  muscles  and  nerves  should  be  infiltrated  with  antitoxin  as 
soon  as  possible  after  an  injury  has  been  received. 


SERUM  TREATMENT  OF  TETANUS  773 

According  to  Mayer  and  Ransom,  Marie  and  Morax,  absorption 
occurs  along  the  axis-cylinders  of  motor  nerves,  the  intramuscular  end- 
ings of  which  the  toxin  penetrates.  The  experiments  of  Field,  Cerno- 
vodeanu,  and  Henni  indicate,  however,  that  the  toxins  are  absorbed  by 
way  of  the  lymphatics  of  the  nerves,  and  not  by  way  of  the  axis-cylinders; 
the  latter  view  is  now  most  generally  accepted. 

Even  though  the  toxin  gains  entrance  to  the  blood,  it  cannot  injure 
the  motor  nerve  tissue  directly,  as,  for  example,  by  means  of  the  blood- 
vessels supplying  the  central  nervous  system.  As  was  previously  stated, 
the  toxin  in  the  blood  and  lymph  channels  may  reach  the  central  nervous 
system  only  in  an  indirect  manner,  through  the  end-plates  or  lymphatics 
of  other  motor  nerves. 

Ascending  centripetally  along  the  motor  plates  and  lymphatics,  the 
poison  reaches  the  motor  spinal  ganglia  on  the  side  inoculated;  it  then 
affects  the  ganglia  on  the  opposite  side,  making  them  hypersensitive. 
The  visible  result  of  this  hypersensitiveness  is  the  highly  increased  muscle 
tonus — i.  6.,  rigidity.  If  the  supply  continues,  the  toxin  next  affects 
the  nearest  sensory  apparatus:  there  is  an  increase  in  the  reflexes,  but 
only  when  the  affected  portion  is  irritated.  In  the  further  course  of  the 
poisoning  the  toxin  as  it  ascends  continues  to  affect  more  and  more 
motor  centers  and  also  the  neighboring  sensory  apparatus.  This  leads 
to  spasm  of  all  the  striated  muscles  and  general  reflex  tetanus  (Park). 

3.  Regardless  of  the  severity  of  the  infection,  there  is  always  an  in- 
cubation period  in  tetanus  during  which  the  bacilli  multiply  and  produce 
toxin  which  is  traveling  toward  the  tissues  of  the  central  nervous  system. 
Antitoxin  in  sufficient  amount  will  neutralize  the  toxin  as  quickly  as  it  is 
produced,  and  thus  protect  the  infected  individual  until  the  leukocytes  and 
other  body-cells  have  destroyed  the  bacilli  and  spores. 

In  general  terms,  the  severer  the  wound  and  the  heavier  the  infec- 
tion, the  shorter  will  be  the  incubation  period  and  the  higher  the  mortal- 
ity. In  acute  tetanus  the  incubation  period  is  less  than  ten  days;  in 
chronic  tetanus  this  period  is  much  longer  and  more  variable. 

The  toxin  is  produced,  and  may  be  absorbed  during  or  at  least  soon  after 
the  first  twenty- four  hours  following  infection;  this  explains  thenecessity  for 
administering  antitoxin  as  soon  as  possible  after  the  injury  hasbeenreceived. 

Action  of  Tetanus  Antitoxin. — 1.  Tetanus  antitoxin  will  neutralize 
free  toxin  in  a  chemical  manner  similar  in  some  respects  to  that  by  which 
neutralization  of  an  acid  by  an  alkali  is  effected.  It  is  generally  believed 
that  as  soon  as  the  molecule  of  antitoxin  has  become  united  with  a  mol- 
ecule of  toxin,  the  latter  is  rendered  inert.  It  may  be  possible,  however, 
for  the  toxin  molecule  to  become  dissociated  and  attack  nerve-cells  or 


774  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

other  molecules  of  antitoxin;  this  is  one  reason  for  the  necessity  of  giv- 
ing large  doses  of  antitoxin  in  order  than  an  excess  may  be  at  hand. 

2.  When  tetanus  toxin  has  once  united  with  nerve-cells,  it  is  difficult 
or  impossible  for  the  antitoxin  to  effect  its  neutralization.     Hence  the 
greatest  value  of  the  antitoxin  lies  in  prophylaxis;    when  properly  ad- 
ministered, however,  it  is  possible  for  the  antitoxin  to  aid  in  the  cure  of 
tetanus,  and  its  use  should  never  be  omitted  in  the  treatment  of  any 
case. 

The  value  of  antitoxin  in  the  treatment  of  tetanus  is  probably  de- 
pendent upon  the  following  two  factors:  (1)  Neutralization  of  all  free 
toxin  as  quickly  as  it  is  secreted  and  before  it  is  absorbed  by  the  nervous 
tissue;  (2)  actual  dissociation  or  neutralization  of  some  of  the  toxiii 
" loosely  united"  with  nerve-cells  or  suspended  in  the  lymph  after  it 
has  left  the  capillaries  and  before  it  is  taken  up  by  the  nerve-cells. 

3.  Aside  from  its  chief  antitoxic  action,  anti-tetanus  serum  probably 
contains  anti-aggressins  or  bacteriotropins  that  aid  phagocytosis  by 
overcoming  their  repelling  or  negatively  chemotactic  influence.     This 
may,  however,  be  accomplished  by  simple  neutralization  of  the  toxins, 
which  impairs  their  leukotoxic  action  sufficiently  to  permit  living  leuko- 
cytes to  engulf  and  destroy  the  bacilli. 

Methods  of  Administering  Tetanus  Antitoxin. — Recent  investiga- 
tions and  case  reports  show  quite  conclusively  that  in  the  treatment  of 
tetanus  as  much  depends  upon  the  method  of  administering  antitoxin 
as  upon  the  quantity  administered. 

1.  Absorption  by  the  subcutaneous  route  is  so  slow  that  it  should  not  be 
depended  upon  in  the  treatment  of  tetanus.     While  it  is  true  that  the  mor- 
tality of  tetanus  has  been  reduced  about  20  per  cent,  by  the  administra- 
tion of  large  amounts  of  serum  by  this  route,  it  should  be  emphasized 
that  a  smaller  amount,  given  subdurally  or  intravenously,  will  yield  even 
better  results.     Knorr  has  shown  experimentally  that  after  subcutaneous 
injection  the  maximum  quantity  of  antitoxin  is  not  found  in  the  blood 
until  twenty-four  hours  have  elapsed.     Since  every  hour  counts  heavily 
in  the  chances  for  recovery  when  symptoms  of  tetanus  have  appeared, 
it  may  be  laid  down  as  a  general  rule  that  the  first  doses  of  antitoxin 
should  be  given  subdurally  or  intravenously.     The  subcutaneous  route 
may  be  chosen  when  serum  is  given  for  prophylactic  purposes  at  the  time 
of  injury,  but  should  not  be  relied  upon  in  the  treatment  of  tetanus. 

2.  Intramuscular  injections  may  be  given  to  keep  up  the  good  effect 
of  antitoxin  after  the  first  doses  have  been  given  subdurally  and  intra- 
venously, and  are  to  be  preferred  to  the  subcutaneous  route  whenever 
the  physician  is  unable  to  inject  the  serum  subdurally  and  intravenously. 


SERUM  TREATMENT  OF  TETANUS  775 

3.  For  the  rapid  neutralization  of  toxin  free  in  the  body-fluids  serum 
should  be  given  intravenously.    In  the  treatment  of  tetanus  from  10,000 
to  20,000  units  may  be  given  by  this  route  as  early  as  possible.     While 
it  is  conceded  that  when  the  toxin  has  become  firmly  bound  to  the  tissues 
of  the  central  nervous  system  dissociation  by  the  use  of  antitoxin  is  not 
possible,  yet  one  can  never  know,  in  a  given  case,  how  firm  this  union 
has  become,  and  clinical  results,  supported  by  animal  experimentation, 
show  that  life  may  be  preserved  by  large  doses  of  antitoxin  injected  into 
the  blood. 

4.  The  experimental  work  of  Pennin,1  Park  and  Nicoll,2  supported 
by  the  clinical  results  reported  by  various  observers,  shows  quite  con- 
clusively that  the  subdural  route  is  a  very  efficacious  and  valuable  avenue 
by  which  to  administer  antitoxin  in  the  treatment  of  tetanus.     The  serum 
should  be  given  by  the  gravity  method,  in  exactly  the  same  manner  as 
in  giving  antimeningitic  serum.     To  insure  its  thorough  dissemination 
throughout  the  spinal  meninges  the  antitoxin  should  be  diluted,  if 
necessary,  with  normal  salt  solution.     As  a  rule,  the  amount  injected 
should  be  slightly  less  than  the  amount  of  fluid  withdrawn.     In  the 
case  of  a  "dry  tap, "  if  the  operator  is  reasonably  sure  of  having  entered 
the  canal,  from  3  to  5  c.c.  of  serum  may  be  injected.     It  is  generally 
necessary  to  repeat  this  injection  within  twenty-four  hours. 

The  reason  for  administering  antitoxin  subdurally  is  apparent  when 
it  is  remembered  that  neither  the  central  nervous  system  nor  the  peri- 
pheral nerves  take  up  antitoxin  direct  from  the  blood  (Park).  Only 
after  very  large  intravenous  doses  are  traces  of  antitoxin  found  in  the 
cerebrospinal  fluid,  and  animals  passively  and  actively  immunized  may 
be  rendered  tetanic  if  the  toxin  is  injected  directly  into  the  central 
nervous  system  or  into  the  nerve.  While  antitoxin  injected  subduraliy 
finally  passes  over  into  the  blood,  it  will  neutralize  any  free  or  dissoci- 
ated toxin  before  the  latter  has  developed  any  harmful  tendency. 

5.  To  neutralize  any  toxin  that  may  have  been  absorbed  by  a  nerve 
it  may  be  advisable  to  inject  antitoxin  directly  into  the  nerve,  and  these 
intraneural  injections  under  anesthesia  are  advised  by  Ashhurst,  John, 
and  others  as  part  of  the  rational  treatment  of  tetanus. 

The  technic  of  these  injections  has  been  described  elsewhere. 

Prophylaxis  of  Tetanus. — The  most  successful  preventive  treatment, 
and  practically  the  only  successful  curative  one  after  the  disease  has  de- 
veloped, is  by  means  of  tetanus  antitoxin.  As  a  prophylactic  remedy 
this  antitoxin  exceeds  in  value  even  diphtheria  antitoxin;  therapeutic- 

1  Mitt.  a.  d.  Grenzgebiet  d.  Med.  und  Chir.,  1913.  xxvii,  1. 

2  Jour.  Amer.  Med.  Assoc.,  1914,  Ixiii,  235. 


776  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

ally,  however,  it  is  far  inferior  to  the  latter,  for  the  reason  that  part  of 
the  toxin  produced  by  the  tetanus  bacilli  soon  unites  with  the  nerve- 
cells  of  the  spinal  cord. 

One  of  the  greatest  dangers  from  this  terrible  infection  lies  in  the 
fact  that  while  the  local  lesion  may  show  no  signs  of  disturbance,  the 
central  nervous  system  may  suddenly  manifest  symptoms  of  poisoning. 
The  wounds  that  are  likely  to  contain  the  tetanus  bacillus  are  the  following: 
All  wounds  that  may  contain  dirt  contaminated  by  manure,  such  as 
that  from  the  streets,  stables,  barns,  and  even  fields;  wounds  made  by 
firecrackers  or  toy  pistols;  gunshot  wounds,  especially  those  made  by 
blank  cartridges;  crushing  injuries,  made  by  machinery  or  in  other  ways. 
The  feet  and  hands  are  especially  prone  to  be  infected  with  tetanus  germs. 
Street  injuries  that  are  not  deep  or  perforating,  but  grinding  and  lacerat- 
ing, are  very  likely  to  develop  tetanus  infection.  It  has  also  been  stated 
that  tetanus  bacilli  may  be  harbored  in  an  old  injury,  and  yet  cause  no 
symptoms  until  some  additional  injury  or  general  disturbance  of  the 
body  causes  the  normal  protection  against  infection  to  be  broken  down, 
when  toxins  from  the  bacilli  may  be  absorbed  and  tetanic  symptoms 
develop.  This  theory  would  seem  to  be  responsible  for  an  otherwise 
apparently  unaccountable  development  of  tetanus. 

From  what  has  been  said  it  will  be  seen  that  any  injuries  received  on 
the  street,  or  those  inflicted  on  workers  about  horses  or  cattle  and  in 
stables,  are  more  likely  to  develop  tetanus  than  are  injuries  received 
in  other  ways.  New-born  babies  may  be  infected  through  the  stump  of 
the  umbilical  cord.  Likewise  a  suppurating  wound,  or  even  a  fresh 
wound,  which  may  be  innocent  at  first,  may  become  infected  with  the 
tetanus  bacillus  if  the  wound  or  suppurating  focus  is  improperly  cared 
for.  Many  cases  of  vaccinal  tetanus  can  thus  be  accounted  for,  i.  e., 
due  to  negligence  in  the  care  and  treatment  of  the  wound.  It  is  now 
generally  agreed  that  proper  treatment  of  the  original  wound,  combined  with 
the  administration  of  tetanus  antitoxin,  will  surely  prevent  the  development 
of  lockjaw, 

In  former  years  Fourth  of  July  wounds  claimed  a  heavy  toll  of  fatal- 
ities due  to  tetanus.  Owing  to  the  efforts  of  the  American  Medical 
Association  municipalities  have  been  urged  to  adopt  legislative  measures 
for  enforcing  a  saner  form  of  celebration,  and  efforts  have  been  made  to 
educate  physicians  in  the  proper  care  of  these  wounds  and  to  impress 
upon  them  the  great  prophylactic  value  of  tetanus  antitoxin.  These 
efforts  have  been  crowned  with  success,  as  statistics  collected  from  all 
parts  of  the  country  will  show.  In  1903  there  were  in  the  United  States 
406  deaths  from  tetanus;  in  1904,  91;  in  1905,  87;  in  1906,  75;  in  1907, 


SERUM  TREATMENT  OF  TETANUS  777 

73;  in  1908,  76;  in  1911,  18  cases  and  10  deaths;  in  1912,  7  cases  with 
6  deaths,  and  in  1913,  4  cases  with  3  deaths. 

While  it  is  not  within  the  province  of  this  chapter  to  deal  with  sur- 
gical technic,  the  proper  cleansing  and  care  of  a  wound  constitute  so 
important  a  part  of  the  prophylaxis  of  tetanus  that  I  shall  refer  to  this 
subject,  quoting  largely  from  the  technic  recently  described  by  Ashhurst 
and  John.1 

Surgical  Treatment. — 1.  The  surrounding  skin  should  be  painted 
with  a  3  per  cent,  alcoholic  solution  of  iodin. 

2.  All  foreign  material  should  be  removed  from  the  wound,  and  to  do 
this  properly  all  parts  of  the  wound  should  be  made  accessible  by  wide 
incision,  under  ether,  if  necessary.     This  is  especially  true  of  a  puncture 
wound.     It  should  then  be  freed  from  all  tags  and  loose  shreds  of  tissue 
by  means  of  the  scissors,  and  the  whole  wound  swabbed  with  the  3  per 
cent,  iodin  solution.     The  wound  should  next  be  dressed  with  gauze 
soaked  in  the  same  solution.     The  use  of  strong  caustics  is  inadvisable, 
as  they  cause  sloughing  and  tend  to  produce  a  good  focus  for  the  growth 
of  tetanus  bacilli. 

3.  The  wound  should  be  dressed  daily  at  first,  being  exposed  and 
thoroughly  irrigated  with  hydrogen  dioxid  solution,  and  then  dressed 
with  the  gauze  saturated  with  the  iodin  solution.     As  soon  as  healthy 
granulations   have    formed,   balsam  of    Peru   applications   should  be 
made. 

4.  Antitetanic  powders  have  been  prepared,  made  up  with  anti- 
septics, and  although  experimentally  their  use  has  seemed  to  be  suc- 
cessful in  preventing  the  development  or  absorption  of  tetanus  toxin, 
still  it  has  not  as  yet  shown  that  these  results  were  not  merely  due  to  the 
strong  antiseptic  that  was  combined  with  the  antitoxin  powder.     It 
might,  however,  be  well  to  apply  tetanus  antitoxin  and  antitetanic 
powder  to  the  open  wound,  but  these  remedies  are  not  to  be  relied  upon  nor 
accepted  as  substitutes  for  the  injection  of  antitoxin. 

Use  of  Antitoxin. — The  prophylactic  use  of  tetanus  antitoxin  has 
not  infrequently  been  unsuccessful,  due  probably  to  the  fact  that  it  was 
used  incorrectly. 

1.  Antitoxin  should  be  given  as  soon  as  possible  after  the  wound  has 
been  inflicted,  and  best  at  the  time  the  primary  treatment  is  given. 
The  antitoxin  should  be  injected  "as  near  the  wound  as  possible,  so  as 
to  flood  the  tissues  in  the  immediate  vicinity, "  and,  if  possible,  it  should 
be  given  intramuscularly,  so  that  the  motor  nerves  may  absorb  it  rapidly. 
1  Amer.  Jour.  Med.  Sci.,  1913,  cxlvi,  No.  1. 


778  '  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

If  any  nerves  are  exposed,  antitoxin  should  be  injected  into  them.  The 
injection  of  1500  units  of  antitoxin  is  generally  advised  as  the  first  prophy- 
lactic dose. 

2.  From  the  fact  that  tetanus  antitoxin  is  one  of  the  albuminous 
constituents  of  horse  serum  that  are  foreign  to  the  human  system,  in  the 
human  being  the  antitoxin  is  rapidly  eliminated  in  from  eight  to  ten 
days  after  the  injection  is  administered.     Knorr  has  found,  as  the  result 
of  animal  experiments,  that  by  the  sixth  day  only  one-third,  and  by  the 
twelfth  day  only  one-fiftieth,  of  the  optimum  quantity  remained  in  the 
blood.     Hence  it  is  important,  if  antitoxin  is  to  prove  useful,  that  it  should 
be  present  in  the  system  for  two  or  three  weeks  after  receipt  of  the  injury, 
especially  as  it  cannot  be  determined  when  the  tetanus  bacillus  first  gained 
access  to  the  wound.     There  should  be  a  second  intramuscular  injection 
of  1000  units  of  antitoxin  about  the  end  of  the  first  week,  and  perhaps  a 
similar  dose  at  the  end  of  the  second  week.     While  certain  individuals  may 
develop  serum  sickness,  no  dangerous  symptoms  have  been  observed 
to  result  from  the  use  of  tetanus  antitoxin. 

3.  If  the  surgeon  is  first  consulted  several  days  after  the  injury  has 
been  inflicted,  the  wound  should  be  opened  and  dressed  as  previously 
described,  and,  in  addition  to  the  intramuscular  injection  of  1500  units 
of  antitoxin  in  the  neighborhood  of  the  wound,  it  will  be  good  practice 
to  inject  an  additional  5000  or  10,000  units  intravenously.     It  requires 
at  least  twenty-four  hours  for  the  antitoxin  to  be  absorbed  from  the 
subcutaneous  tissues,  and  immediate  neutralization  of  any  toxin  present 
in  the  blood  may  mean  a  great  deal  from  the  standpoint  of  prognosis 
if  tetanus  should  develop. 

Treatment  of  Tetanus. — While  the  great  value  of  antitetanic  serum 
as  a  preventive  is  unquestioned,  as  a  specific  cure  it  has  fallen  short  of 
the  earliest  expectations.  It  has  been  shown  experimentally,  however, 
that  tetanus  antitoxin  may  save  the  lives  of  animals  already  manifesting 
the  symptoms  of  an  otherwise  fatal  intoxication.  In  order  to  accomplish 
this  result  the  serum  must  be  given  in  doses  several  hundred  times  the 
size  required  merely  for  protective  purposes,  and  it  must  be  injected 
within  a  short  time — from  twenty-four  to  thirty-six  hours — after  the 
onset  of  the  tetanus.  Furthermore,  statistics  favor  the  use  of  the  serum 
as  the  mortality  has  been  reduced  from  80  to  85  per  cent,  to  60  or  65  per 
cent,  in  cases  receiving  serum  treatment. 

The  recognition  of  the  natural  limitations  of  the  serum  treatment  of 
tetanus  will  serve  to  emphasize  the  importance  of  its  proper  adminis- 
tration. A  large  number  of  units  must  be  given,  and  must  be  injected 


SERUM  TREATMENT  OF  TETANUS  779 

in  places  best  suited  to  secure  the  maximum  neutralizing  action  of  the 
serum. 

Surgical  Treatment. — 1.  The  site  of  the  wound  should  be  located, 
and  if  possible  incised  under  ether  or  chloroform  anesthesia  and  thor- 
oughly cleansed  of  foreign  material  and  necrotic  tissue.  It  should  then 
be  swabbed  with  the  3  per  cent,  alcoholic  solution  of  iodin,  washed  with 
hydrogen  dioxid  solution,  and  packed  loosely  with  gauze  soaked  in  the 
iodin  solution. 

2.  Cauterization  with  pure  phenol,  followed  by  alcohol,  may  be  em- 
ployed, but,  as  a  rule,  the  weaker  germicide  is  preferable  in  order  not  to 
produce  necrosis  of  the  tissues,  which  furnishes  pabulum  for  bacterial 
growth.  The  wound  should  be  dressed  daily. 

Serum  Treatment. — A  maximum  amount  of  antitoxin  should  be 
given  the  patient  as  soon  as  possible,  and  the  greater  the  delay  in  giving 
the  antitoxin,  the  greater  is  the  amount  required.  Since  absorption 
after  subcutaneous  injection  is  very  slow,  valuable  time  may  be  lost, 
and  since  enormous  amounts  must  be  given,  at  great  expense,  this  route 
possesses  much  less  value  than  the  intravenous  and  intraspinal  methods. 

1.  Administer  intravenously  from  10,000  to  20,000  units  of  antitoxin 
at  once,  and  repeat  the  dose  if  no  effect  is  apparent  or  if  the  good  effect 
wears  off  in  about  from  eighteen  to  twenty-four  hours  (the  technic  is 
described  on  p.  742).     After  one  or  two  intravenous  injections  the  good 
effect  may  be  maintained  by  direct  intramuscular  injections  of  from  5000 
to  10,000  units  for  one  or  two  doses. 

2.  From  3000  to  5000  units  should  be  given  intraspinally  by  means 
of  lumbar  puncture.     This  dose  should  be  repeated  every  twenty-four 
hours  unless  the  symptoms  have  markedly  ameliorated.     The  technic 
of  this  injection  is  described  on  p.  746.     A  quantity  of  cerebrospinal 
fluid  should  be  removed  before  the  serum  is  injected.     After  the  first 
injection  the  fluid  may  be  found  to  have  become  cloudy,  with  a  large 
increase  of  cells,  especially  of  the  polynuclear  variety,  although  bac- 
teriologically  it  may  be  sterile.     This  outpouring  of  leukocytes  is  prob- 
ably a  reaction  to  the  irritant  effect  of  the  serum,  and  especially  of  the 
preservative  it  contains. 

3.  If  a  surgeon  is  at  hand,  from  500  to  1000  units  of  antitoxin  should 
be  injected  slowly  intravenously  into  the  sheaths  of  the  nerve-trunks 
leading  from  the  infected  region.     These  injections  are  directed  to  be 
made  as  near  the  trunk  as  possible,  and  to  distend  the  nerve  so  as  partly 
to  neutralize  and  partly  mechanically  to  interrupt  the  passage  of  toxin 
to  the  cord  or  brain. 


780  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

Both  the  intraspinal  and  the  intraneural  injections  are  given  under 
light  chloroform  anesthesia. 

General  Treatment. — A  large  number  of  substances  have  been  ad- 
vocated  in  the  treatment  of  tetanus;  of  these,  the  most  common  are 
injections  of  phenol  and  intraspinal  injections  of  magnesium  sulphate. 
While  phenol  may  be  well  tolerated  by  tetanus  patients,  Ashhurst  and 
John  believe  that  all  these  treatments  are  of  little  value,  and  that  spinal 
injections  of  magnesium  sulphate  are  dangerous. 

1.  Chloral  hydrate  and  potassium  bromid  should  be  given  by  mouth 
or  by  rectum,  in  sufficient  quantities  to  produce  sleep  and  quiet.     Drugs, 
such  as  those  of  antagonistic  physiologic  activity,  are  more  or  less  suc- 
cessful and  frequently  of  aid  when  given  in  conjunction  with  the  anti- 
toxin. 

2.  While  combating  the  disease,  the  general  care  of  the  patient  should 
not  be  forgotten.     A  purgative  should  be  administered  early.     Simple, 
nourishing,  non-stimulating  food  should  be  given  by  the  mouth,  if 
possible,  or  by  the  nasal  tube,  if  necessary.     Absolute  quiet  should  be 
maintained.     Distention  of  the  bladder  from  retention  of  urine  should 
be  guarded  against.     If  water  is  not  well  absorbed,  and  especially  if 
there  is  peritoneal  or  pelvic  inflammation,  saline  injections  into  the  colon 
should  be  given. 

Results  of  the  Antitoxin  Treatment  of  Tetanus. — As  was  previously 
stated,  the  prophylactic  value  of  tetanus  antitoxin  has  been  proved  beyond 
any  reasonable  doubt.  This  does  not  imply,  however,  that  the  simple  intro- 
duction of  1000  units  of  antitoxin  beneath  the  skin  will  surely  protect 
the  patient,  as  the  percentage  of  cases  developing  tetanus  even  after  the 
serum  has  been  given  is  altogether  too  high.  As  was  pointed  out  under 
Prophylactic  Treatment,  the  wound  must  receive  thorough  surgical  at- 
tention, and  the  antitoxin  must  be  injected  in  such  places  where  it  will 
have  the  greatest  opportunity  to  neutralize  the  toxin.  Even  if  tetanus 
should  develop  under  these  conditions,  it  is  likely  to  be  mild  and  the 
prognosis  would  be  much  more  favorable. 

Tetanus  antitoxin  has  likewise  been  very  successful  in  veterinary 
practice,  especially  after  castration  and  other  operations,  in  injuries,  and 
among  horses  used  for  the  purpose  of  producing  diphtheria  antitoxin 
and  other  immune  serums. 

While  the  curative  value  of  tetanus  antitoxin  has  not  come  up  to  expecta- 
tions, more  recent  carefully  prepared  statistics  indicate  that,  with  serum 
treatment,  the  mortality  is  reduced  at  least  20  per  cent.  This  includes  cases 
treated  by  the  subcutaneous  injection  of  antitoxin,  and  it  must  be  em- 


SERUM  TREATMENT  OF  TETANUS  781 

phasized  that,  in  order  to  secure  the  best  results,  tetanus  must  be  treated 
in  a  rational  manner  according  to  its  pathology.  Under  these  circum- 
stances we  can  confidently  expect  a  greater  reduction  in  mortality.  But 
at  any  rate  no  physician  should  withhold  antitoxin  in  the  treatment  if 
there  are  any  possible  means  of  obtaining  it.  If  only  3000  units  may  be 
had,  it  is  far  better  to  inject  this  amount  intraspinally  than  subcu- 
taneously. 

Pennin,1  in  a  recent  and  thorough  review,  reaches  the  general  con- 
clusion that  anti-tetanus  serum  has  reduced  the  mortality  of  tetanus 
approximately  20  per  cent.  He  gives  figures  from  Denmark  that  are 
especially  valuable,  because  they  were  gathered  from  a  small  area,  and 
hence  represent  fairly  uniform  conditions:  Of  199  cases  not  receiving 
serum,  only  21  per  cent,  recovered;  whereas  of  189  cases  treated  with 
serum,  42.3  per  cent,  recovered.  Of  92  acute  cases  with  an  incubation 
period  of  less  than  ten  days  24.2  per  cent,  recovered  when  serum  was 
used,  whereas  of  94  cases  treated  without  serum  only  5.3  per  cent,  re- 
covered. It  is  significant  that  these  Danish  figures  correspond  closely 
to  the  American  statistics  and  those  of  other  countries.  Irons2  has 
recently  tabulated  the  results  of  225  cases  of  tetanus  treated  with  anti- 
toxin collected  from  hospitals  and  private  records  for  the  years  1907  to 
1913.  The  mortality  in  all  cases  receiving  serum  was  61.77  per  cent.; 
in  21  cases  without  serum  the  mortality  was  85.7  per  cent.  The  latter 
figures  correspond  quite  closely  with  the  general  mortality  of  about  85 
per  cent,  of  tetanus  treated  without  serum.  Irons'  figures  also  show  the 
influence  of  large  doses  of  antitoxin;  of  57  cases  receiving  a  small  dose 
of  antitoxin  (3000  units  or  less  subcutaneously),  the  mortality  was  73.7 
per  cent.;  of  143  cases  receiving  large  doses  (over  3000  units  subcu- 
taneously, or  3000  or  less  intraspinally  or  intravenously),  the  mortality 
was  57.3  per  cent.  Magnesium  sulphate  was  given  intraspinally  in  18 
cases  which  also  received  serum;  in  4  cases — 2  acute  and  2  chronic — 
the  patients  recovered,  giving  a  mortality  for  the  group  of  77  per  cent. 
In  2  cases  death  recurred  shortly  after  injection  with  symptoms  of  re- 
spiratory failure. 

In  view  of  this  evidence  in  favor  of  antitoxin  in  the  treatment  of 
tetanus  it  is  apparent  that  the  physician  is  compelled  to  give  every 
patient  with  tetanus  the  opportunity  to  obtain  this  20  per  cent,  or  more 
benefit  by  administering  the  serum  promptly  and  correctly. 

1  Mitt.  a.  d.  Grenzgeb.  d.  Med.  u.  Chir.,  1913,  xxvii. 

2  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  20,  25. 


782  PASSIVE   IMMUNIZATION — SERUM    THERAPY 

SERUM  TREATMENT  OF  DYSENTERY 

Soon  after  the  discovery  of  a  bacillus  of  dysentery  by  Shiga  in  1892 
the  treatment  of  bacillary  dysentery  by  the  use  of  immune  serums  was 
undertaken.  Following  the  discovery  of  the  etiologic  importance  of 
Shiga's  bacillus  in  the  dysenteries  of  Asiatic  countries,  similar  investiga- 
tions were  made  in  various  parts  of  the  world  and  various  bacilli  were 
isolated.  At  first  these  microorganisms  were  all  regarded  as  being  iden- 
tical, but  further  investigation  has  shown  that  marked  differences  are 
apparent,  and  two  main  types  are  now  recognized:  one  type  (Shiga) 
does  not  ferment  mannite  and  produces  a  soluble  or  extracellular  toxin, 
and  a  second  type  (Flexner-Harris,  Hissy,  etc.)  ferments  mannite  and 
does  not  produce  an  extracellular  toxin.  A  further  discussion  of  these 
bacilli  will  be  found  in  Chapter  VII. 

Dysentery  caused  by  the  bacilli  of  the  Kruse-Shiga  type  may  be  re- 
garded as  a  form  of  intoxication  analogous  to  the  intoxication  of  diph- 
theria. The  intestine,  where  the  bacilli  lodge,  corresponds  to  the  throat, 
which  is  the  site  of  infection  in  diphtheria;  here  the  bacilli  develop  and 
produce  their  toxins,  and  these  toxins,  when  absorbed  into  the  circula- 
tion, in  turn  produce  the  symptoms  of  the  disease. 

Antitoxin  has  been  prepared  for  the  bacilli  of  the  Kruse-Shiga  type, 
and  these  have  yielded  fairly  satisfactory  results  in  the  prophylaxis  and 
cure  of  this  variety  of  bacillary  dysentery.  Antiserums  for  the  mannite- 
fermenting  group  of  bacilli  (Flexner,  Harris,  Hess,  Duval,  etc.)  have  not 
proved  of  much  value  in  the  treatment  of  these  infections.  Bacilli 
of  the  latter  group  are  largely  responsible  for  the  dysenteries  in  this 
country,  and  also  for  a  percentage  of  cases  of  ileocolitis  of  infancy. 
Since  the  antiserum  of  the  Shiga  bacillus  is  of  practically  no  value  in  the 
treatment  of  infections  caused  by  bacilli  of  other  groups,  the  serum 
treatment  of  dysentery  is  employed  mainly  in  European  and  Asiatic 
countries,  where  infections  with  this  group  are  common.  After  fairly 
extensive  trials  in  this  country  the  serum  treatment  of  infantile  diarrheas 
and  true  dysenteries  has  proved  disappointing. 

The  preparation  and  standardization  of  dysentery  antitoxin  is  de- 
scribed in  Chapter  XIV. 

Administration  and  Uses. — Dysentery  antitoxin  has  been  used  both 
in  the  prophylaxis  and  in  the  cure  of  this  infection.  The  doses  of  serum 
advised  by  various  observers  vary  considerably,  owing  to  the  marked 
differences  that  exist  in  the  potency  of  these  serums.  Since  the  various 
manufacturers  do  not  employ  the  same  standards,  the  physician  should 
use  the  serum  in  accordance  with  the  printed  directions  that  accom- 
pany each  package. 


SERUM   TREATMENT    OF   DYSENTERY  783 

For  prophylactic  purposes,  usually  from  10  to  20  c.c.  are  recommended, 
and  it  is  further  advised  to  repeat  the  injection  after  two  or  three  weeks, 
as  the  protection  lasts  only  a  short  time. 

For  curative  purposes,  Shiga  has  advised  10  c.c.  for  mild  cases  and 
from  20  to  60  c.c.  for  severer  cases.  It  may  be  necessary  to  repeat  the 
injections  several  times.  Vaillard  and  Doyle  have  given  as  much  as 
from  80  to  100  c.c.,  and  have  repeated  this  dose  on  the  following  days. 
When  the  serum  is  being  used  during  an  epidemic,  it  is  advisable  to  as- 
certain beforehand  the  nature  of  the  infection,  as  the  antiserum  for  the  Shiga 
bacillus  is  highly  specific  and  is  not  likely  to  prove  of  value  in  the  treatment 
of  infections  caused  by  the  Flexner  type  of  bacillus.  Otherwise  a  poly- 
valent serum  should  be  used. 

The  injections  have  usually  been  given  subcutaneously.  Better 
results  would,  no  doubt,  be  obtained  in  the  treatment  of  severe  infec- 
tions by  administering  large  doses  of  serum  intravenously. 

Results. — Adequate  statistics  regarding  the  value  of  the  serum  in 
the  prophylaxis  and  treatment  of  dysentery  are  not  yet  available.  From 
the  prophylactic  standpoint,  encouraging  results  have  been  reported  by 
Kruse,  Shiga,  Vaillard  and  Dopter,  Rosculet,  and  others,  and  it  would 
appear  that  passive  immunization  is  of  value  in  combating  localized 
outbreaks,  such  as  occur  in  institutions  and  armies. 

From  the  curative  standpoint,  most  observers  agree  that  the  use  of  a 
potent  serum  will  reduce  the  mortality  of  acute  cases  at  least  from  30 
to  50  per  cent.  Shiga  reports  a  drop  in  the  mortality  in  Japan  of  from 
22  to  26  per  cent,  to  9  to  12  per  cent. ;  Kruse  obtained  a  reduction  in 
mortality  of  about  10  per  cent.  Vaillard  and  Dopter 1  treated  96  cases, 
with  but  1  death;  Rosenthal2  treated  157  cases  with  7  deaths, — a  mor- 
tality of  4.5  per  cent,  as  compared  with  that  of  10  to  11  per  cent,  oc- 
curring in  other  German  hospitals.  Coyne  and  Auche3  treated  11  cases 
due  to  the  Flexner  type  of  bacillus  and  report  good  results.  Ruffer 
and  Willmore,4  Violle,5  Rogers,6  Brau7  and  Bahr8  have  also  reported 
favorably  upon  the  use  of  the  serum.  A  highly  potent  and  polyvalent 
serum  should  be  in  readiness  during  war,  and  particularly  in  camps 
and  hospitals.  In  a  thorough  investigation  made  in  the  United  States  in 
1903  by  the  Rockefeller  Institute,  under  the  direction  of  Flexner,  it  was 

1  Ann.  Inst.  Past.,  1906,  xx,  321;  1907,  xxi,  241. 
^Deut.  med.  Wchnschr.,  1904,  xxx,  No.  19. 

3  Compt.  rend.  Soc.  Biol.,  1906,  Ix,  No.  26. 

4  Brit.  Med.  Jour.,  1908,  11,  1176;  ibid.,  1909,  ii,  462. 

5  Arch.  med.  et  pharm.  nav.,  1912,  xcviii,  61. 

6  Dysenteries,  Their  Differentiation  and  Treatment,  London,  1913,  290. 

7  Ann.  d.  hyg.  et  meU,  1913,  xvi,  710.  8  Brit.  Med.  Jour.,  1914. 


784  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

found  that  the  results  of  the  serum  treatment  of  ileocolitis,  among  chil- 
dren at  least,  were  quite  disappointing.  This  is  largely  due  to  the  fact 
that  several  different  strains  of  bacilli  may  be  the  cause  of  an  infection, 
and  unless  a  corresponding  antiserum  is  employed  for  the  particular  type 
causing  the  infection  in  a  given  case,  good  results  cannot  be  expected. 
Probably  if  some  means  were  discovered  for  making  a  prompt  bacterio- 
logic  diagnosis,  and  if  several  immune  serums  were  on  hand  for  the  treat- 
ment of  infections  caused  by  the  main  types,  after  the  methods  worked 
out  by  Neufeld,  Dochez,  and  Cole  in  the  treatment  of  pneumonia,  good 
results  may  be  obtained. 

The  curative  effect  of  dysentery  antitoxin  is  shown  by  a  reduction  in 
the  number  of  stools,  by  the  fact  that  blood  and  pus  disappear  from  the 
discharges,  pain  and  tenesmus  are  relieved,  the  temperature  becomes 
normal,  and  the  patient  gains  in  weight.  Individual  observers  are  fre- 
quently enthusiastic  over  the  results  obtained  in  individual  cases,  and 
no  doubt  these  are  striking  in  those  instances  where  the  antiserum  ap- 
pears to  be  specific  for  the  particular  form  of  infection. 


THE  SERUM  TREATMENT  OF  HOG  CHOLERA 

While  the  cause  of  hog  cholera  has  not  as  yet  been  discovered,  it  is  a 
well-known  fact  that  the  virus  is  present  in  the  blood  of  infected  animals, 
and  it  is  possible,  by  immunizing  healthy  hogs  with  gradually  increasing 
doses  of  infected  blood,  to  prepare  a  potent  immune  serum  that  will 
prove  of  value  in  the  prophylaxis  and  cure  of  hog  cholera.  The  nature 
of  this  serum  is  unknown.  It  possesses  many  of  the  features  of  an  anti- 
toxin, and  for  the  present  may  be  classed  with  these. 

Production  and  Standardization  of  Hog  Cholera  Serum. — Healthy  hogs  weigh- 
ing about  100  pounds  are  selected,  and  injected  subcutaneously  with  40  c.c.  of 
hog  cholera  serum  per  hundred  pounds  of  weight.  Two  or  three  days  following 
this  protecting  dose  they  are  injected  intravenously  with  3  or  4  c.c.  of  sterile,  defibrin- 
ated  blood,  obtained  from  an  animal  suffering  from  the  disease;  or  the  animals 
may  be  exposed  in  pens  known  to  be  infected.  If  the  animals  live  for  one  month 
without  showing  evidences  of  toxemia,  they  receive  another  injection  of  5  c.c.  of 
infected  blood  (virus).  Two  or  three  weeks  later  they  receive  another  injection  of 
from  15  to  20  c.c.  of  infected  blood;  in  from  fifteen  to  twenty-one  days  after  this 
inoculation  they  receive  a  final  injection  of  from  4  to  5  c.c.  of  virus  per  pound  of 
body  weight.  Animals  tolerating  the  last  injection  are  said  to  be  hyperimmune,  and 
are  bled  in  ten  days.  In  hyperimmunizing  the  animal  some  prefer  to  inject  the  virus 
intraperitoneally  instead  of  intravenously.  If  this  method  is  adopted,  about  double 
the  dose  of  infected  blood  (virus)  is  required.  About  5  per  cent,  of  animals 
succumb  during  the  period  of  immunization. 


THE    SERUM   TREATMENT   OF   HOG    CHOLERA  785 

The  immunized  hogs  are  bled  aseptically  from  the  tail  by  snipping  off  the  tip, 
and  5  c.c.  of  blood  per  pound  of  weight  is  collected  in  sterile  vessels.  Each  animal  is 
bled  once  a  week  until  three  or  four  bleedings  have  been  made. 

In  about  one  week  after  the  last  bleeding  they  are  hyperimmunized  again  by 
giving  them  4  or  5  c.c.  of  infected  blood  per  pound  of  body  weight,  and  additional 
bleedings  are  made  so  long  as  the  tail  lasts. 

The  serums  secured  in  the  several  bleedings  are  mixed  together.  The  third  or 
fourth  bleeding  is  said  to  be  most  potent  or  to  contain  the  greatest  number  of  anti- 
bodies. 

In  testing  and  standardizing  the  immune  serum,  six  hogs,  weighing  about  100 
pounds  each,  are  placed  in  an  infected  pen.  No.  1  receives  no  serum  and  is  a  control; 
No.  2  receives  15  c.c.  of  serum;  No!  3  receives  20  c.c.;  No.  4  receives  30  c.c.;  No.  5 
receives  35  c.c.;  and  No.  6  receives  40  c.c.  of  immune  serum.  The  animals  are 
allowed  to  remain  in  the  pens  until  the  control  succumbs  and  the  protecting  dose  of 
serum  has  been  determined.  In  this  manner  we  can  determine  just  about  the  amount 
of  serum  required  to  protect  100  pounds  of  hog.  The  Pennsylvania  Live  Stock 
Sanitary  Board  has  found  that  it  does  not  require  quite  40  c.c.  of  serum,  but  this 
amount  is  recommended  for  safety,  and  because  the  weight  may  not  be  judged 
accurately. 

Hog-cholera  serum  is  used  in  both  the  prophylaxis  and  the  thera- 
peutic management  of  this  disease.  For  prophylactic  purposes  for  each 
100  pounds  of  hog  40  c.c.  of  serum  are  injected.  These  injections  are 
usually  given  subcutaneously  and  occasionally  intramuscularly.  In 
some  instances  active  and  passive  immunization  is  practised  by  the 
simultaneous  injection  of  2  c.c.  of  virus,  together  with  40  c.c.  of  immune 
serum  per  100  pounds  of  weight.  Owing  to  the  danger  of  spreading  the 
disease,  this  method  is  not  generally  employed. 

The  results  of  prophylactic  immunization  are  excellent,  and  the 
method  is  valuable  in  checking  epidemics.  Immunity  is  said  to  last  for 
six  months  after  vaccination. 

For  curative  purposes  the  serum  has  yielded  good  results,  providing 
it  is  administered  not  later  than  the  fourth  day  after  the  animal  shows 
evidences  of  the  disease.  Several  injections  may  be  required,  and  the 
intramuscular  route  should  be  chosen,  because  quicker  absorption  is 
thus  insured. 

Calf  cholera  serum  has  been  prepared  by  immunizing  horses 
with  strains  of  colon,  paracolon,  and  other  bacilli  belonging  to  these 
groups,  isolated  from  calves  dying  of  calf  cholera.  The  immune  serum 
should  agglutinate  the  microorganisms  used  in  its  production  in  dilu- 
tions of  1:2000  to  1:500. 

This  serum  has  proved  of  value  in  the  prevention  of  calf  cholera, 
and  may  be  of  benefit  in  the  treatment,  providing  that  it  is  prepared 
with  the  same  strain  or  strains  of  microorganisms  responsible  for  the 
infection. 
50 


786  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

SERUM  TREATMENT  OF  SNAKE-BITES 

The  nature  of  snake  venom  is  discussed  in  Chapter  VII,  and  the 
method  of  preparing  antivenomous  serum  is  described  in  Chapter  XIV. 
Calmette's  antivenene  for  cobra  venom  is  useful  in  the  treatment  of 
cobra  envenomation,  but  is  not  serviceable  for  the  treatment  of  other 
snake-bites,  as  shown  by  Martin  for  Australian  serpents  and  by  McFar- 
land  for  American  snakes.  In  the  venoms  of  our  snakes,  as,  for  example, 
the  rattlesnake,  copper-head,  and  moccasin,  the  poison  is  essentially 
locally  destructive,  the  respiratory  poison  being  of  secondary  impor- 
tance. McFarland  failed  to  immunize  horses  against  this  locally  de- 
structive poison.  Later  Noguchi  and  Madsden  succeeded  in  producing 
an  antiserum,  prepared  by  immunizing  horses  with  venom  after  the 
toxophorous  groups  of  the  molecules  had  been  destroyed,  capable  of 
neutralizing  the  hemorrhagin  of  the  Crotalus  venom. 

The  serums  of  Calmette,  Noguchi,  and  others  are  useful  in  the  treat- 
ment of  their  respective  envenomations,  but  aside  from  India,  Brazil, 
and  a  few  other  reptile-infested  countries,  as  well  as  in  zoological  gardens 
and  laboratories  where  snakes  are  kept,  the  serums  have  a  very  limited 
sphere  of  usefulness. 

SERUM  TREATMENT  OF  HAY-FEVER 

The  nature  of  pollen  toxin  has  been  discussed  in  Chapter  VII,  and  the 
preparation  of  antitoxin  in  Chapter  XIV. 

Dunbar  has  prepared  an  antitoxin  for  certain  pollen;  this  may  be 
obtained  commercially.  The  method  of  administration  consists  in 
dropping  the  serum  into  the  eyes  and  sniffing  it  into  the  nose  at  the  onset 
of  an  attack.  It  is  necessary  for  the  patient  to  carry  the  serum  and 
dropper  about,  as  the  effects  produced  are  of  short  duration,  and  the 
patient  is  subject  to  repeated  reinfections.  Subcutaneous  injections 
are  not  advisable,  as  considerable  local  edema  is  produced,  and  the 
amount  of  protection  afforded  is  uncertain. 

Many  observers,  as,  for  example,  Semon,1  McBride,2  Knight,3 
Throst,4  Weichardt,5  and  others,  report  that  the  serum  affords  a  tem- 
porary relief  which  is  grateful  to  the  patient,  but  which  cannot  be  said 
to  be  curative  of  the  disease.  In  some  instances  it  fails  altogether,  and 
in  these  it  is  reasonable  to  assume  that  the  antitoxin  has  not  been  pre- 
pared from  the  particular  pollen  that  infected  the  patient.  An  intoler- 
ance to  the  serum  may  be  excited. 

1  Brit.  Med.  Jour.,  1903,  ii,  123,  220.  2  Edin.  Med.  Jour.,  1903,  ii,  7. 

3  Med.  Record,  March  10,  1906.     , 

4  Mtinch.  med.  Wochenschr.,  June  9,  1903. 

5  Centralbl.  f .  Bakt.,  Abst.,  1906,  xxxviii,  493. 


ANTIBACTERIAL    IMMUNIZATION  787 

More  recently  the  possibilities  of  effecting  active  immunization 
against  hay-fever  have  been  shown  by  Claves1  with  the  pollen  of  rag- 
weed. The  method  is  still  in  the  experimental  stage,  but  it  is  reasonable 
to  assume  that  vaccines  may  be  prepared  for  the  pollen  of  various  plants 
usually  responsible  for  the  hay-fever  and  autumnal  catarrhs  of  this 
country.  (See  page  708.) 

ANTIBACTERIAL  IMMUNIZATION 

General  Considerations. — It  may  be  stated  that  antibacterial  serums 
have  not  been  found  of  equal  value  to  the  antitoxins, .  either  in  the 
prophylaxis  or  in  the  treatment  of  their  respective  infections.  It  is 
true,  however,  that  antimeningococcus  serum  has  reduced  the  mortality 
of  epidemic  cerebrospinal  meningitis  from  75  to  90  per  cent,  to  30  per 
cent,  and  less,  and  has  thereby  firmly  established  its  value  in  the  treat- 
ment of  this  dreaded  infection.  Recent  work  in  pneumonia  has  de- 
veloped a  method  of  serum  therapy  that  has  proved  of  value  in  the  treat- 
ment of  this  disease,  and  it  is  likewise  true  that  antistreptococcus  and 
antigonococcus  serums  yield  at  times  and  in  individual  cases  most 
prompt  and  happy  results.  But  the  expectations  for  serum  therapy 
that  were  aroused  in  1894  with  the  discovery  of  diphtheria  antitoxin 
have  not  been  fully  realized,  although  at  the  present  time  the  reasons 
for  failure  are  being  studied,  understood,  and  gradually  overcome. 

Granting  that  serum  therapy  could  be  reduced  to  the  simple  prop- 
osition of  bringing  specific  antibodies  into  relation  with  the  micro- 
organisms producing  a  given  infection,  the  process  remains  quite  intri- 
cate, largely  owing  to  the  fact  that  although  different  strains  of  the  same 
microorganism  may  possess  identical  morphologic  and  biologic  charac- 
teristics, yet  they  vary  not  only  in  pathogenicity,  but  also  in  the  speci- 
ficity of  the  antibodies  that  they  stimulate  the  body-cells  to  produce. 
In  other  words,  serum  therapy  is  more  specific  than  it  is  generally  con- 
sidered to  be.  For  instance,  the  antibodies  of  one  strain  of  pneumococcus 
may  have  little  or  no  action  upon  another  strain,  and  the  same  is  prob- 
ably true  of  the  various  pathogenic  bacilli  and  groups  of  streptococci, 
gonococci,  and  to  a  lesser  extent  also  of  meningococci.  This  fact  has 
long  been  known,  and  an  effort  has  been  made  to  overcome  the  difficulty 
by  immunizing  horses  with  a  large  number  of  different  strains  of  the 
same  microorganism  in  the  hope  that  the  polyvalent  serum  so  produced 
would  contain  sufficient  antibodies  for  all  or  most  infections  of  the 
various  strains  of  the  particular  microorganisms  in  question.  With 
diphtheria  and  tetanus  bacilli,  the  soluble  toxin  is  apparently  quite 
1  Proc.  Soc.  Exper.  Biol.  and  Med.,  1913,  x,  69. 


788  PASSIVE    IMMUNIZATION SERUM    THERAPY 

similar  for  all  strains,  so  that  immunization  with  the  toxin  of  one  yields 
an  antitoxin  capable  of  neutralizing  the  toxins  of  all.  With  dysentery 
bacilli,  snake  venoms,  and  pollens,  however,  the  toxins  are  more  specific, 
and  produce  more  specific  antitoxins. 

Recent  work  in  pneumonia  by  Cole  and  Dochez  and  their  coworkers 
in  the  Rockefeller  Institute,  and  Neufeld  in  Germany,  indicates  a  more 
promising  future  for  antibacterial  immunization.  These  investigators 
have  been  able  to  divide  pneumococci  into  four  main  groups,  have 
worked  out  a  relatively  quick  method  of  determining  the  group  to  which 
a  particular  pneumococcus  belongs,  and  have  prepared  immune  serums 
for  these  main  groups.  By  injecting  the  serum  corresponding  to  the 
strain  producing  a  given  infection,  encouraging  results  have  been  ob- 
tained in  the  treatment  of  pneumonia,  whereas  the  polyvalent  serums 
have  been  found,  after  quite  extensive  use,  to  yield  indifferent  results, 
due  in  part  to  a  relatively  low  content  in  the  particular  antibodies  for  that 
certain  strain  causing  a  given  infection. 

These  investigations  in  pneumonia  are  of  great  importance  because 
they  reveal  an  immense  field  of  interesting  and  similar  researches  in 
streptococcus,  gonococcus,  meningococcus,  typhoid,  and  other  infections. 
While  it  is  obviously  impossible  to  prepare  an  immune  serum  for  each 
and  every  strain  of  microorganism,  it  may  be  possible  to  subdivide 
strains  into  a  few  main  groups  and  then  discover  a  method  for  quickly 
determining  to  which  group  a  particular  culture  belongs,  so  that  the 
corresponding  immune  serum  may  be  administered.  In  this  manner 
we  may  be  able  to  reduce  the  30  per  cent,  mortality  still  remaining  in 
epidemic  meningitis  and  otherwise  place  the  treatment  of  specific  in- 
fections upon  a  more  strictly  scientific  basis,  as  in  the  serum  treatment 
of  diphtheria. 

As  previously  stated,  the  method  and  route  of  injecting  an  immune 
serum  are  of  considerable  importance  in  serum  therapy.  Large  doses  of 
serum  should  be  given  until  the  desired  effect  is  secured,  or  until  it 
becomes  evident  that  more  can  be  produced.  In  the  mean  time  manu- 
facturers should  make  every  effort  to  produce  potent  serums  and  to 
concentrate  them,  if  possible,  just  as  diphtheria  antitoxin  is  concentrated. 


THE  SERUM  TREATMENT  OF  MENINGOCOCCUS  MENINGITIS 
During  the  pandemic  of  meningococcal  cerebrospinal  meningitis  in 
1904-05  several  laboratories  sought  to  produce  an  immune  serum  for  the 
purpose  of  treating  human  cases  of  this  infection. 


THE  SERUM  TREATMENT  OF  MENINGOCOCCUS  MENINGITIS    789 

After  pursuing  experimental  studies  on  the  subject  on  the  lower 
animals,  Jochmann,1  in  1905,  immunized  a  horse  and  used  the  serum  in 
the  treatment  of  38  cases  of  epidemic  meningitis.  At  first  he  employed 
the  subcutaneous  method  of  injection,  and  later  he  used  the  intraspinal 
method.  The  results  were  quite  encouraging,  and  during  the  following 
year  30  more  cases  were  treated,  with  a  resulting  mortality  of  27  per 
cent.,  as  against  a  mortality  of  53  per  cent,  in  untreated  cases. 

At  about  the  same  time  Kolle  and  Wassermann2  reported  that  they 
had  prepared  an  antimeningococcus  serum,  which  had  not,  however, 
up  to  that  time  been  used  in  the  treatment  of  human  infections.  A  year 
later  serum  was  administered  subcutaneously  and  then  intraspinally, 
with  encouraging  results. 

In  this  country  Park  had,  in  1905,  prepared  an  antimeningococcus 
serum,  which  was  used  in  the  treatment  of  20  cases  in  Hartford,  Conn., 
by  subcutaneous  injection,  but  without  beneficial  results.  Jochmann 
had,  in  the  mean  time,  shown  the  superiority  of  intraspinal  injections, 
and  this  method  soon  supplanted  the  subcutaneous  method. 

In  1905  Flexner  began  a  series  of  studies  regarding  experimental 
meningococcus  infections  in  the  lower  animals,  and  the  therapeutic 
value  of  antimeningococcus  serum.  These  valuable  experiments  at- 
tracted the  attention  of  the  world,  and  placed  this  method  of  treatment 
upon  a  firm  basis.  In  1906  Flexner3  proved  that  a  specific  immune 
antimeningococcus  serum  could  be  produced  that,  if  injected  intra- 
spinally, would  save  the  lives  of  monkeys.  Later  horses  were  immunized 
and  the  serum  used  in  the  treatment  of  human  infections  during  an 
epidemic  in  Akron,  Ohio,  in  May,  1907.  In  a  short  time  the  serum  was 
used  extensively  in  other  epidemics,  and  a  report  of  these  early  cases  was 
made  by  Flexner4  in  1907.  A  later  report  by  Flexner  and  Jobling,5 
covering  the  treatment  of  400  cases,  showed  that  the  mortality  had  been 
reduced  from  75  per  cent,  to  below  30  per  cent.  In  1909  they  reported6 
upon  712  cases  treated  with  the  serum,  with  a  mortality  of  31.4  per  cent. 
In  a  more  recent  report  Flexner7  reviewed  all  the  cases — 1300  in  number 
—gathered  from  all  parts  of  the  world,  treated  with  serum  prepared  in 
the  Rockefeller  Institute.  The  general  mortality  rate  is  given  as  30.9 
per  cent.,  as  against  75  to  80  per  cent,  among  cases  not  receiving  serum 

1  Deut.  med.  Wochenschr.,  1906,  xxii,  No.  20,  788. 

2  Deut.  med.  Wochenschr.,  1906,  xxxii,  No.  16. 

3  Jour.  Amer.  Med.  Assoc.,  1906,  xlvii,  560. 

4  Jcur.  Exper.  Med.,  1907,  ix,  168.  B  Jour.  Exper.  Med.,  1908,  x,  141. 

6  Jour.  Amer.  Med.  Assoc.,  1909,  liii,  1443. 

7  Jour.  Exper.  Med.,  1913,  xvii,  553. 


790  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

treatment.  Of  1394  cases  treated  with  serum  during  the  Texas  epi- 
demic1 (1912),  the  mortality  was  37  per  cent.,  as  compared  with  a  mor- 
tality of  77  per  cent,  among  562  cases  receiving  no  serum. 

Good  results  have  been  reported  by  many  observers  with  Joch- 
mann's,  Kolle  and  Wassermann's,  RuppePs,  Paltauf  s,  and  Dopter's 
serums,  and  the  serums  have  been  prepared  by  several  commercial 
biologic  laboratories,  so  that  the  curative  value  of  antimeningococcus 
serum  is  definitely  established. 

Nature  of  Meningococcus  Meningitis. — Bacteriologic  and  patho- 
logic evidence  indicates  that  the  first  stage  of  meningococcus  meningitis 
consists  of  a  meningococcus  bacteremia,  the  virulent  meningococci 
gaining  access  to  the  blood-stream  through  the  upper  air-passages. 
Later  the  infection  becomes  localized  in  the  spinal  and  cerebral  meninges. 
It  is  probable  that  the  microorganism  causes  a  primary  nasopharyn- 
gitis,  and  in  some  instances  the  meninges  may  be  infected  by  direct 
extension  through  the  sphenoid  and  ethmoid  sinuses.  The  main  symp- 
toms and  lesions  of  the  disease,  and  several  of  the  complications,  for 
example,  the  paralyses,  eye  complications,  deafness,  hydrocephalus, 
and  mental  disturbances,  are  probably  directly  due  to  suppuration  of 
the  meninges,  with  involvement  of  accessory  and  motor  nerve-roots, 
meningeal  irritation,  and  pressure  from  the  accumulation  of  exudate 
in  the  ventricles  and  subarachnoid  space.  Complications,  such  as 
arthritis,  pyelitis,  endocarditis,  adenitis,  etc.,  are  due  to  the  bacteremia, 
which  may  become  chronic  and  be  accompanied  by  deposits  of  menin- 
gococci in  the  various  tissues  and  organs.  In  addition  to  these  com- 
plications there  is  probably  a  varying  degree  of  general  toxemia,  due  to  a 
soluble  toxin  or  endotoxin  liberated  through  lysis  of  the  cocci. 

Treatment  of  Epidemic  Meningitis. — Although  cerebrospinal  menin- 
gitis may  be  considered  primarily  as  a  general  infection,  in  the  majority 
of  instances  local  suppuration  of  the  meninges  constitutes  the  main 
lesion.  For  anatomic  and  physiologic  reasons,  however,  it  is  impossible 
to  treat  the  disease  according  to  the  ordinary  principles  governing  the 
treatment  of  localized  suppuration,  as,  for  example,  by  continuous 
drainage  and  by  cleansing  the  affected  parts  with  germicidal  solutions. 
Unaided,  the  leukocyt.es  and  body-fluids  are  generally  unable  to  destroy 
the  cocci  and  terminate  the  infection  before  serious  harm  to  important 
nerves  and  nerve-centers  has  resulted,  so  that  epidemic  meningitis,  with 
a  mortality  of  from  75  to  90  per  cent.,  and  followed  by  more  or  less 

1  Sophian:  Epidemic  Cerebrospinal  Meningitis,  St.  Louis,  1913.  Report  of  Dr. 
Steiner,  President  of  the  Texas  State  Board  of  Health. 


THE  SERUM  TREATMENT  OF  MENINGOCOCCUS  MENINGITIS    791 

serious  sequelae,  which  few  survive,  is  one  of  the  most  dreaded  of  in- 
fections. 

In  administering  antimeningitic  serum  we  aim  to  assist  the  patient's 
leukocytes  and  body-fluids  to  overcome  the  infection.  Repeated  spinal 
punctures  remove  portions  of  the  infective  material  mechanically,  but 
the  greatest  dependence  in  bringing  about  quick  destruction  of  the 
cocci  and  effecting  recovery  of  the  patient  is  to  be  placed  upon  the  serum. 


Preparation  of  the  Antimeningococcus  Serum. — Method  of  Flexner  and  Jobling. — 
1.  Many  strains  of  meningococcus  are  used  in  order  that  a  polyvalent  serum  may 
be  prepared.  Fresh  strains  from  new  epidemics  and  sporadic  cases  are  constantly 
added.  ''Fast "  strains,  or  those  isolated  from  cases  in  which  the  serum  has  produced  noj 
beneficial  effect,  are  especially  desirable.  By  means  of  complement-fixation  tests 
Olmstead1  and  her  associates  have  been  able  to  classify  meningococci  into  two  main 
groups;  similar  results  have  been  secured  by  Kitchens  and  Robinson2  with  a  protec- 
tive test.  Both  tests  are  apparently  highly  specific  and  serve  to  differentiate  menin- 
gococci from  similar  microorganisms. 

2.  Immunization  is  performed  first  with  an  autolysate  of  the  meningococci,  and 
later  with  living  cultures. 

3.  Stock  cultures  are  kept  alive  by  transplanting  them  every  four  days  in  slants 
of  ascitic  glucose  agar,  neutral  to  phenolphthalein. 

4.  In  preparing  the  autolysate,  the  cultures  are  subcultured  first  on  glucose-agar 
slants  without  serum.    After  twenty-four  hours'  growth  about  3  c.c.  of  salt  solution 
are  added  to  each  slant,  and  the  culture  emulsified.    One  cubic  centimeter  is  then 
poured  over  the  surface  of  glucose-agar  slants  in  large  500  c.c.  Blake  bottles.    After 
twenty-four  hours'  incubation  heavy,  uniform,  and  diffuse  growths  are  secured. 

Add  10  c.c.  of  normal  salt  solution  to  each  bottle  and  wash  off  the  culture.  If 
necessary,  a  long  heavy  platinum  loop  may  be  used.  Each  bottle  is  tested  for  con- 
tamination by  staining  a  smear  according  to  the  method  of  Gram.  Each  bottle  is 
emptied  into  a  common  vessel;  2  per  cent,  toluol  is  added,  mixed  well,  and  incubated 
for  from  eighteen  to  twenty-four  hours.  The  toluol  is  then  allowed  to  evaporate,  or 
it  may  be  immediately  filtered  off  through  sterile  gauze  saturated  with  salt  solution. 
The  preparation  is  kept  in  a  refrigerator  and  should  be  prepared  fresh  every  month. 

5.  The  injections  are  given  subcutaneously  about  the  neck  and  abdomen.    Young 
and  healthy  horses  are  selected  for  the  purpose.    The  first  dose  consists  of  2  c.c.  of 
the  autolysate,  and  this  is  gradually  increased,  depending  on  the  manner  in  which  the 
animal  reacts,  until  10  c.c.  are  given  in  a  single  dose.    Then  inject  2  c.c.  of  living  cul- 
ture diluted  with  two  parts  of  salt  solution,  and  increase  the  doses,  the  same  as  with 
the  autolysate,  until  10  c.c.  of  culture  are  given  at  one  injection.    Next  inject  living 
cultures  and  autolysate  alternately,  until  a  maximum  of  from  30  to  35  c.c.  are  given 
in  one  dose;    this  last  is  then  used  as  the  constant  dose  until  the  immunization  has 
been  completed. 

Injections  are  given  every  five  to  seven  days  until  the  large  doses  are  reached, 
when  they  are  given  every  ten  days  or  two  weeks. 

Horses  usually  show  quite  marked  reactions,  such  as  fever,  depression,  and 
induration  about  the  site  of  injection,  but  if  due  care  is  exercised,  few  animals  are 
lost  during  the  immunization. 

6.  Bleedings  are  usually  begun  about  the  fourth  month  after  immunization  has 
been  instituted.     The  horses  are  bled  aseptically  from  a  jugular  vein  about  every 

1  Jour.  Immunology,  1916,  1,  307.  2  Jour.  Immunology,  1916,  1,  345. 


792  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

two  weeks,  from  six  to  eight  liters  of  blood  being  removed  at  each  sitting.  The  serum 
is  separated  and  preserved  with  trikresol.  Recent  investigations  indicate  that  trikresol 
may  be  partly  responsible  for  paralysis  of  the  respiratory  centers,  and  at  present  every 
effort  should  be  made  to  collect  and  market  the  serum  in  a  strictly  aseptic  manner,  so  that 
none  or  but  very  little  preservative  is  required.1  Efforts  are  being  made  at  present  to 
discover  an  efficient  volatile  antiseptic  that  may  be  driven  off  by  warming  the  serum 
at  body  temperature. 

Rapid  Method  of  Amoss  and  Wollstein.2 — These  investigators  have  recently  advo- 
cated a  rapid  method  of  immunization  consisting  in  inoculating  alternately  several 
strains  of  living  meningococci  and  parameningococci  and  the  autolyzed  products 
of  each,  by  which  a  potent  polyvalent  serum  may  be  produced  in  eight  to  twelve 
weeks  instead  of  in  the  ten  months  required  by  the  subcutaneous  method.  In  the 
preparation  of  a  polyvalent  serum  a  twenty-four-hour  agar  slant  culture  of  meningo- 
cocci is  removed  with  2  c.c.  of  salt  solution  and  0.1  c.c.  suspended  in  15  c.c.  of  salt 
solution  and  injected  very  slowly  into  the  circulation  of  a  horse.  The  temperature  is 
taken  hourly  and  should  not  rise  over  3°  C.;  twenty-four  hours  later  0.2  c.c.  of  sus- 
pension, and  on  the  third  day  0.3  c.c.  are  given.  After  the  lapse  of  seven  days  a 
series  of  three  injections  of  living  parameningococci  are  given  in  doses  of  about 
0.3  c.c.  of  the  emulsion  or  sufficient  to  give  a  temperature  reaction  of  about  2.5°  to 
3°  C.  After  a  lapse  of  seven  days  three  intravenous  injections  of  an  autolysate 
composed  of  equal  parts  of  autolysate  of  meningococci  and  parameningococci  are 
given.  A  series  of  three  injections  of  living  meningococci  and  parameningococci 
follow  in  doses  which  may  be  run  up  to  0.6  c.c.  of  emulsion,  with  every  third  series 
consisting  of  injections  of  the  mixed  autoly sates;  after  ten  to  twelve  weeks  a  potent 
serum  is  generally  produced.  In  order  to  make  the  serum  as  polyvalent  as  possible 
a  large  number  of  strains  of  meningococci  and  parameningococci  should  be  used. 

Severe  reactions  due  to  hypersensitiveness  of  the  horse  to  the  meningococci  or 
its  products  are  especially  likely  in  the  first  dose  of  each  series  after  three  or  four 
series  of  injections  have  been  given.  In  order  to  desensitize,  a  portion  of  the  first 
injection  of  each  series  is  injected  intravenously,  and  two  hours  later  the  remainder 
of  the  dose  is  given.  Danger  due  to  agglutinated  cocci  is  lessened  by  diluting  the 
dose  in  15  to  20  c.c.  of  salt  solution  and  injecting  very  slowly. 

Standardization  of  Antimeningococcus  Serum. — An  accurate  method  of  stand- 
ardizing antimeningococcus  serum  has  not  as  yet  been  devised.  In  the  selection  of  a 
serum  physicians  must,  therefore,  be  guided  by  the  reputation  of  the  manufacturers. 

An  antimeningococcic  serum  of  high  antibody  content  has  antitoxic,  bacterio- 
tropic,  and  bactericidal  properties.  Kraus  and  Dorr  consider  that  the  chief  function 
of  the  serum  is  antitoxic;  Flexner  and  Jobling,  Neufeld,  Jochmann,  and  Wassermann 
believe  that  its  bacteriotropic  properties  are  its  most  important  qualities. 

The  following  methods  for  testing  an  immune  serum  are  in  use  or  have  been 
advocated;  none  of  them  is,  however,  sufficiently  reliable  to  serve  as  a  definite 
measure  of  antibody  content  or  of  curative  value;  two  of  them,  the  bacteriotropic  and 
the  complement-fixation  test,  are  most  widely  used  in  laboratories  for  the  purpose  of  estimat- 
ing the  antibody  content  of  a  serum. 

1.  Bacteriotropic  Titration. — While  the  antimeningitic  serum  was  being  prepared 
at  the  Rockefeller  Institute,  Jobling*  used  the  opsonic  test  in  standardization  as  the 
method  of  choice,  on  account  of  the  part  taken  by  specific  opsonins  in  promoting 
recovery  from  meningococcus  infections.  As  a  definite  and  suitable  standard  of 
strength  Jobling  has  suggested  that  a  serum  be  accepted  as  satisfactory  when  it  shows 

1  Hall,  W.:   Bull.  No.  91,  Hyg.  Lab.,  U.  S.  P.  H.  S.,  1914. 

2  Jour.  Exper.  Med.,  1916,  23,  403.      3  Jour.  Exper.  Med.,  1909,  xi,  6,  4. 


THE    SERUM   TREATMENT   OF   MENINGOCOCCUS    MENINGITIS        793 

unmistakable  phagocytic  activity  in  dilutions  up  to  1 : 5000.   The  method  of  Neuf eld 
is  used,  as  described  on  page  203. 

2.  Complement-fixation  Tests. — The  advantages  of  these  tests  are  that  the  same 
polyvalent  antigen  may  be  used  as  is  employed  for  purposes  of  immunization;  the 
technic  is  simple,  and  the  reactions  are  usually  sharp  and  definite.  According  to 
Sophian,  in  a  series  of  comparisons  with  opsonic  and  complement-fixation  tests  the 
results  corresponded  in  every  instance,  a  high  opsonic  content  being  accompanied  by 
a  high  complement-fixation  reading.  The  latter  indicates,  at  least,  that  the  horse  has 
responded  to  immunization  and  that  curative  antibodies  probably  are  present.  Labor- 
atories usually  adopt  their  own  standards  in  preparing  antimeningococcic  serum.  In 
the  complement-fixation  test  Kolle  requires  complete  inhibition  of  hemolysis  with 
0.1  c.c.  of  serum. 

Kitchens  and  Hansen1  have  advocated  a  dried  meningococcus  antigen  prepared 
as  follows: 

1.  The  cultures  are  grown  on  salt-free  agar  at  37°  C.  for  sixteen  to  eighteen  hours. 

2.  The  growth  is  collected  and  suspended  in  distilled  water;  about  10  c.c.  dis- 
tilled water  being  used  for  each  20  square  inches  of  agar  surface. 

3.  To  this  suspension  is  added  an  equal  volume  of  95  per  cent,  ethyl  alcohol. 

4.  This  mixture  is  immediately  centrifugalized. 

5.  After  the  supernatant  fluid  has  been  removed  the  precipitated  bacteria  are 
again  suspended  in  95  per  cent,  ethyl  alcohol. 

6.  This  process  of  centrifugalization  and  resuspension  is  repeated  a  second  and 
a  third  time,  but  with  ethyl  ether  instead  of  alcohol,  the  original  volume  of  the 
suspension  being  reduced  one-half  each  time. 

7.  The  ether  adhering  to  the  bacteria  after  the  precipitation  and  after  removal 
of  the  supernatant  ether,  is  removed  by  vacuum. 

8.  The  bacterial  mass  now  constituting  the  antigen  is  further  dried  over  phos- 
phorus pentoxid  in  vacuo  for  three  days. 

9.  The  antigen  is  stored  in  tubes — about  0.05  gram  to  each — and  kept  in  vacuo 
over  phosphorus  pentoxid. 

For  use,  a  small  amount  of  the  powder,  0.02  gram,  is  carefully  ground  in  a  mortar, 
20  c.c.  of  normal  salt  solution  being  gradually  added. 
The  technic  of  these  tests  is  given  on  page  524. 

3.  Agglutination  Tests. — These  tests  are  readily  conducted  with  the  polyvalent 
antigen  used  in  immunization,  a  macroscopic  technic,  as  that  described  on  page  301, 
being  employed.    Kolle  regards  a  serum  as  satisfactory  when  it  causes  agglutination 
of  meningococci  in  dilutions. up  to  1:  5000. 

4.  Animal  Inoculation  Tests. — These  tests  have  been  found  quite  irregular  and 
impracticable  for  general  use.    As  stated  by  Jobling,  not  only  does  the  pathogenicity 
of  the  meningococcus  vary  considerably  from  day  to  day,  but  the  resistance  of  animals 
to  this  microorganism  is  also  quite  variable.    By  preparing  a  large  quantity  of  bac- 
terial emulsions  and  using  sufficient  controls  to  determine  the  fatal  dose,  and  by 
employing  a  standard  lethal  dose  of  emulsions  mixed  with  varying  quantities  of 
serum  injected  intraperitoneally  into  250-gram  guinea-pigs,  some  conception  of  the 
protective  value  of  a  serum  may  be  obtained. 

Hitchens  and  Robinson2  have  recently  perfected  an  animal  inoculation  test  which 
promises  the  best  means  for  testing  the  potency  and  standardizing  a  serum.  The 
M.  L.  D.  of  mixed  cultures  of  meningococci  is  determined  by  washing  off  sixteen- 
hour  growths  of  each  culture  on  slants  of  serum-dextrose  agar  with  1  c.c.  of  a  1 : 4 
dilution  of  fresh  guinea-pig  serum.  The  emultions  are  mixed  and  0.5,  0.25,  0.12, 
0.06,  and  0.03  c.c.  injected  intraperitoneally  into  white  mice.  The  M.  L.  D.  is  the 
smallest  lethal  dose  at  the  end  of  forty-eight  hours.  In  testing  a  serum  0.5  c.c.  vary- 

1  Jour.  Immunology,  1916,  1,  355.  2  Jour.  Immunology,  1916,  1,  345. 


794  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

ing  dilutions  are  injected  intraperitoneally  two  hours  before  the  injection  of  1  M.  L.  D. 
culture.  These  protection  tests  have  been  found  to  parallel  the  extent  of  immuni- 
zation more  closely  than  agglutination  or  complement-fixation  tests. 

Action  of  Antimeningococcus  Serum. — As  previously  stated,  ex- 
periments in  vitro  show  that  a  potent  antimeningitic  serum  possesses 
three  chief  antibodies  upon  which  its  curative  powers  probably  depend, 
namely:  (1)  Bacteriotropins  (immune  opsonins),  which  lower  the  re- 
sistance of  the  meningococci  and  facilitate  their  phagocytosis;  (2) 
bactericidins,  which  kill  the  cocci  extracellularly,  either  with  or  without 
final  lysis;  and  (3)  antitoxins,  which  neutralize  the  true  extracellular 
toxin,  which  some  strains  of  meningococci  apparently  produce  in  varying 
degree.  Other  than  these  are  the  agglutinins,  which  probably  aid  in 
bacteriolysis,  and  anti-aggressins,  which  may  assist  in  the  process  of 
phagocytosis. 

Microscopic  examination  of  a  direct  stained  smear  of  the  sediment  of 
cerebrospinal  fluid  obtained  from  fresh  cases  will  show  large  numbers  of 
polynuclear  leukocytes  and  cocci,  the  majority  of  the  latter  being  ex- 
tracellular. As  the  case  improves,  whether  under  serum  treatment  or 
spontaneously,  the  microorganisms  diminish  in  number  and  become 
intracellular,  frequently  appearing  clumped  and  failing  to  grow  in 
culture.  It  would  appear,  therefore,  that  a  cure  is  brought  about 
partly  by  means  of  phagocytosis  aided  by  bacteriotropins;  by  bacte- 
riolysis through  the  agency  of  specific  bacteriolytic  amboceptors  in  the 
immune  serum  and  complements  in  the  spinal  fluid  and  blood-serum,  and 
to  some  extent  by  neutralization  of  a  toxin  with  antitoxin. 

A  potent  antimeningococcus  serum  furnishes  these  main  antibodies, 
and  since  the  first  two  must  act  locally  upon  the  cocci  infecting  the 
meninges,  the  serum  must  be  applied  locally  and  directly  by  intra- 
spinous  and  subdural  injection,  since  only  traces  of  immune  serum  could 
eventually  find  their  way  into  the  cerebrospinal  fluid  if  the  serum  were 
injected  subcutaneously  or  intravenously.  On  the  other  hand,  in  the 
treatment  of  meningococcus  bacteremia  and  toxemia  the  serum  should 
be  injected  intravenously  and  subcutaneously. 

Unfortunately,  an  immune  serum  may  not  contain  the  antibodies 
for  the  cocci  producing  a  given  infection,  and  hence  the  serum,  even 
though  it  is  skilfully  administered  in  large  doses,  will  have  no  influence 
upon  the  disease.  Apparently  the  cocci  of  these  resistant  or  "fast" 
strains  are  uninjured  by  the  antibodies  in  the  serum.  To  overcome  this 
difficulty,  a  large  number  of  different  strains  of  meningococci  are  used 
in  immunizing  horses.  If,  however,  the  serum  of  one  laboratory  is 


THE    SERUM   TREATMENT    OF   MENINGOCOCCUS    MENINGITIS        795 

found  to  exert  no  beneficial  effect,  the  physician  should  use  the  serum 
of  another,  for  different  laboratories  probably  immunize  their  horses 
with  cultures  not  in  common  use.  It  is  highly  desirable  to  secure  cultures 
of  these  "fast"  strains.  These  should  be  sent  at  once  to  laboratories  engaged 
in  the  production  of  antimeningitic  serum,  for  the  larger  the  number  of  these 
strains  used  in  immunization,  the  more  potent  and  valuable  will  the  serum 
be. 

Administration  and  Dosage  of  Antimeningitic  Serum. — In  Acute 
Meningitis. — As  a  rule,  serum  should  be  injected  into  the  spinal  canal  as 
early  in  the  disease  as  possible,  and  in  such  maximum  amount  as  is  com- 
patible with  safety.  Intraspinal  injection  is  absolutely  necessary,  for 
the  serum  must  be  brought  into  contact  with  the  infected  membranes, 
and  only  a  trace  would  reach  the  spinal  fluid  if  the  serum  were  injected 
subcutaneously  or  intravenously.  The  advantages  of  early  administra- 
tion are  obvious,  and  if  the  symptoms  are  indefinite,  the  physician 
should  not  hesitate  to  perform  lumbar  puncture  and  to  secure  fluid  for 
microscopic  examination,  just  as  he  would  take  a  throat  or  nose  culture 
to  aid  in  the  diagnosis  of  diphtheria.  The  maximum,  or  at  least  an 
adequate,  amount  of  serum  should  be  injected,  care  being  observed  to 
avoid  undue  pressure  as  a  result  of  injecting  too  quickly  or  too  large  an 
amount.  This  administration  of  antimeningitic  serum  is,  therefore,  an 
important  and  delicate,  though  relatively  simple,  procedure. 

1.  The  technic  of  intraspinal  injection  has  been  described  on  p.  746. 
Whenever  possible,  the  serum  should  be  injected  by  the  gravity  method, 
and  the  amounts  of  fluid  withdrawn  and  serum  injected  controlled  by 
blood-pressure  readings. 

2.  Lumbar  puncture  is  performed,  and  the  fluid  collected  in  gradu- 
ated tubes.     In  the  ordinary  case,  fluid  may  be  allowed  to  drain  until 
the  blood-pressure  drops  about  10  mm.  of  mercury,  or  if  the  pressure  is 
unchanged  or  rises,  until  the  fluid  flows  about  one  drop  every  three 
seconds,  provided  there  are  no  other  evidences  of  collapse,  such  as  faint- 
ness,  headache,  and  great  restlessness. 

3.  As  a  rule,  the  amount  of  serum  injected  should  be  slightly  less 
than  the  amount  of  fluid  withdrawn.     When  the  injection  is  controlled 
by  the  blood-pressure  readings, — the  amount  varies  considerably, — usu- 
ally the  injection  should  stop  when  the  pressure  falls  another  10  or  15 
mm.     For  adults,  the  dose  of  serum  should  be  about  30  c.c.;    for  an 
infant,  about  15  c.c. 

The  serum  should  be  allowed  to  flow  in  slowly,  an  ordinary  injec- 
tion consuming  at  least  ten  or  fifteen  minutes.  If  symptoms  of  col- 
lapse should  appear  before  an  adequate  amount  of  serum  has  been 


796  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

injected,  the  funnel  may  be  lowered  and  the  spinal  fluids  allowed  to 
flow  out.  When  the  symptoms  have  disappeared,  the  injections  may  be 
continued  and  satisfactorily  completed. 

3.  When  the  physician  cannot  administer  the  serum  by  the  gravity 
method  or  under  blood-pressure  control,  the  injection  may  be  given  by 
means  of  a  syringe  (see  p.  752).  It  should  be  given  slowly,  and  the 
patient  observed  closely  in  order  to  detect  the  general  symptoms  of 
collapse.  The  amount  of  serum  injected  should  not  be  larger  than  the 
amount  of  cerebrospinal  fluid  withdrawn.  According  to  Sophian,  the 
average  doses  are  as  follows: 

DOSE  OF  ANTIMENINGITIC     AMOUNT  OF  FLUID 
SERUM  WITHDRAWN 

1  to  5  years 3  to  12  c.c  12  c.c. 

5  to  10  years 5  to  15  c.c.  15  c.c 

10  to  15  years 10  to  20  c.c.  20  c.c. 

15  to  20  years 15  to  25  c.c.  30  c.c. 

20  years  and  over 20  to  30  c.c.  40  c.c. 

The  injection  of  too  large  a  dose  of  serum  may  be  followed  by  head- 
ache, pain  in  the  back  and  legs,  and  restlessness.  When  the  amount  of 
serum  injected  exceeds  the  amount  of  spinal  fluid  withdrawn  the  symp- 
toms just  named  must  be  regarded  as  the  signal  to  stop;  otherwise  they 
may  be  disregarded. 

Intravenous  Injection. — During  epidemics  of  meningitis  it  may  be 
possible  to  detect  cases  in  the  bacteremic  stage  when  meningococci  are 
present  in  the  blood  and  clear  fluid  is  collecting  in  the  ventricles.  In 
these  and  in  all  severe  fulminant  infections  it  is  good  practice  to  inject 
from  30  to  100  c.c.  of  serum  intramuscularly  or  intravenously.  It  is 
advisable  to  secure  a  culture  of  blood  in  ascites  dextrose  broth  in  all 
cases  in  adults  and  older  children.  If  sufficient  serum  may  be  obtained 
and  the  expense  is  a  secondary  consideration,  an  intravenous  or  intra- 
muscular injection,  given  at  the  outset  and  once  or  twice  during  the  acute 
stage,  may  benefit  the  patient  by  neutralizing  toxins  and  possibly  pre- 
vent complications  due  to  the  entrance  of  meningococci  into  the  blood- 
stream. Furthermore,  as  shown  by  Flexner  and  Amos,1  in  poliomyelitis 
the  intraspinal  injection  of  serum  increases  the  permeability  of  the  cho- 
roid  plexus  and  meninges,  permitting  the  passage  of  immunity  principles 
from  the  blood  into  the  cerebrospinal  fluid.  For  this  reason  the  intra- 
venous or  intramuscular  injection  of  anti meningitis  serum  in  conjunc- 
tion with  intraspinal  injections  may  be  particularly  advantageous  in 
the  treatment  of  severe  infections. 

1  Jour.  Exper.  Med.,  1917,  xxv,  499  and  525. 


THE    SERUM   TREATMENT    OF    MENINGOCOCCUS    MENINGITIS        797 

Repeating  Doses  of  Serum. — It  is  the  general  rule  to  give  an  intra- 
spinal  injection  of  serum  every  day  for  four  days,  and  then  on  alternate 
days  until  the  acute  symptoms  have  subsided,  and  to  resume  the  treat- 
ment if  an  exacerbation  or  a  relapse  occurs.  In  severe  fulminant  in- 
fections, and  especially  if  the  exudate  is  thick  and  only  small  amounts  of 
serum  can  be  introduced  under  pressure,  it  is  well  to  repeat  the  injection 
every  twelve  hours  until  several  doses  have  been  given.  Some  cases 
require  daily  consecutive  injections  for  six  or  more  days;  the  average 
case  will  require  from  four  to  six  injections  if  the  treatment  is  begun 
during  the  acute  stage;  in  the  subacute  and  chronic  cases  many  more 
treatments  are  required.  There  are  two  main  indications  and  guides: 

1.  The  condition  of  the  cerebrospinal  fluid. 

2.  The  clinical  condition  of  the  patient. 

1.  In  most  instances  the  cerebrospinal  fluid  tends  to  clear  up  macro- 
scopically  as  the  disease  improves.     This  is,   however,   occasionally 
misleading,  as  the  fluid  may  become  more  turbid  as  the  result  of  an 
aseptic  meningitis  or  excitation  of  a  polynuclear  leukocytosis-  due  to  the 
serum,  while  in  reality  the  numbers  of  meningococci  are  diminishing 
and  the  patient  is  improving. 

More  accurate  information  is  obtained  by  the  microscopic  examina- 
tion of  a  stained  smear  of  the  sediment  of  the  cerebrospinal  fluid  with- 
drawn. In  fresh  acute  cases  the  cocci  are  numerous  and  mostly  ex- 
tracellular; improvement  is  indicated  by  a  diminution  in  their  number, 
and  by  the  fact  that  they  are  mostly  intracellular.  I  generally  determine 
the  phagocytic  index  or  the  relative  proportion  of  leukocytes  that  have 
engulfed  cocci  and  the  opsonic  index  or  the  relative  number  of  cocci  per 
leukocyte  as  determined  by  counting  a  large  number.  When  many  cocci 
are  present,  or  if  they  are  few  in  number  but  mostly  extracellular,  the 
indications  are  to  puncture  next  day,  even  if  the  clinical  condition  of  the 
patient  is  good  and  the  temperature  is  lower.  The  number  and  position 
of  cocci  are,  therefore,  of  more  importance  as  a  guide  to  subsequent 
injections  than  is  the  total  number  of  pus-cells. 

2.  As  an  indication  for  repeating  the  doses  of  serum  the  clinical  con- 
dition is  of  most  value  when  combined  with  the  examination  of  the 
cerebrospinal  fluid.     Occasionally  the  patient's  condition  may  seem  to 
improve,  although  the  fluid  may  show  numerous  cocci,  which  will  sub- 
sequently aggravate  the  clinical  condition  unless  the  serum  is  adminis- 
tered.    In  favorable  cases  there  is  usually  a  lower  temperature  the  day 
following  an  injection,  and  frequently  delirium  becomes  less  marked  and 
there  is  some  return  to  consciousness.     The  complexion,  which  is  often 


798  PASSIVE    IMMUNIZATION SERUM    THERAPY 

cyanosed  at  first,  regains  a  healthy  color;  the  pain  in  the  head,  neck, 
and  limbs  becomes  less  severe,  although  the  neck  and  spine  may  remain 
stiff  for  several  days.  Finally  the  mind  becomes  clear  and  the  patient 
is  cheerful,  and  no  longer  irritable,  apathetic,  and  hypersensitive.  He 
feels  better  and  his  appetite  returns.  When  this  favorable  outcome 
supervenes,  the  serum  injections  may  be  discontinued,  to  be  resumed, 
however,  upon  the  first  evidence  of  a  relapse.  The  physician  should  be 
on  his  guard  for  the  appearance  of  acute  hydrocephalus,  which  condition 
is  relieved  by  repeated  lumbar  puncture. 

The  Serum  Treatment  of  Cases  with  Thick  Elastic  Exudate. — In  very 
severe  cases  the  exudate  may  be  so  thick  that  it  will  not  flow  from  the 
needle.  In  these  cases  the  serum  should  be  injected  in  small  doses  under 
pressure,  and  the  injections  repeated  every  eight  to  twelve  hours.  As 
they  are  likely  in  any  case  to  terminate  fatally,  the  physician  is  justified 
in  taking  the  risk  of  increasing  intracranial  pressure.  It  may  be  well 
carefully  to  inject  a  small  amount  of  warm  sterile  salt  solution,  which 
will  dilute  the  exudate  and  possibly  start  a  flow;  or  a  second  needle  may 
be  inserted  higher  up,  when  a'  thinner  exudate  is  found,  or  washing  may 
be  possible  by  injecting  salt  solution  in  the  upper  needle  and  draining 
through  the  lower. 

The  Serum  Treatment  of  Cases  with  a  Dry  Canal  and  Cases  of  Posterior 
Basal  Meningitis. — Occasionally  a  patient  improves  clinically  and  the 
amount  of  cerebrospinal  fluid  becomes  very  scanty,  the  spinal  tap  being 
dry,  although  it  is  certain  that  the  needle  has  entered  the  subarachnoid 
space.  In  such  instances  a  small  amount  of  serum  may  be  injected,  or  the 
injection  may  be  dispensed  with  if  the  clinical  condition  continues  to 
improve.  If,  however,  cases  with  dry  canals  present  evidences  of 
toxemia  and  general  aggravation  of  symptoms,  small  doses  of  serum 
should  be  injected  under  pressure  and  the  injections  repeated  as  often  as 
necessary.  The  physician  must  be  very  cautious,  however,  for  if  there 
are  clinical  evidences  of  severe  intracranial  pressure,  it  is  probable  that 
there  is  an  encapsulation  'of  fluid  within  the  ventricles,  and  shutting  off 
of  the  communication  between  the  ventricles  and  the  subarachnoid 
space.  In  this  posterior  basic  meningitis  intraspinal  injections  are 
dangerous  and  aggravate  the  process.  In  infants  it  is  necessary  to  punc- 
ture the  ventricles  through  the  anterior  fontanel  and  in  older  children 
and  adults  by  trephining  at  Kocher's  point,  removing  the  fluid,  and 
if  it  is  found  to  be  cloudy  or  purulent,  injecting  serum.  It  may  be 
necessary  to  tap  both  ventricles  alternately  at  intervals  of  several  days, 
depending  upon  the  reaccumulation  of  fluid  and  pressure  symptoms. 


THE    SERUM   TREATMENT   OF   MENINGOCOCCUS   MENINGITIS       799 

Permanent  drainage  may  be  instituted  in  severe  cases  by  means  of  small 
catheters.  While  the  operation  is  not  usually  dangerous,  the  ultimate 
prognosis  is  very  unfavorable. 

The  Serum  Treatment  of  Subacute  and  Chronic  Meningitis. — If  there 
is  no  evidence  of  sepsis;  if  the  mind  is  clear  and  the  neck  limber;  if  the 
general  conditions  are  good  and  the  cerebrospinal  fluid  is  practically 
cleared  up,  the  affection  is  most  likely  hydrocephalitic,  and  may  be 
relieved  by  repeated  spinal  punctures,  with  removal  of  as  much  fluid  as 
is  safe,  using  blood-pressure  as  an  index.  If  meningococci  are  present 
in  cultures  of  the  fluid,  small  amounts  of  serum  may  be  injected.  The 
prognosis  in  these  cases,  however,  is  generally  bad,  as  the  process  is  pro- 
longed and  the  patient  finally  succumbs. 

In  the  second  form  of  chronic  meningitis,  when  the  meningeal 
symptoms  are  active,  intensified,  and  persistent,  serum  should  be  ad- 
ministered every  few  days  in  the  same  manner  and  in  the  same  dosage 
as  in  the  acute  cases.  Improvement  is,  however,  usually  temporary, 
and  the  ultimate  prognosis  is  very  grave. 

Serum  Sickness. — Intraspinal  injections  of  serum  result  in  the  sen- 
sitization  of  the  patient  in  just  the  same  manner  as  if  serum  were  in- 
jected by  other  routes.  The  percentage  of  cases  developing  serum  sickness 
is  likely  to  be  high,  since  antimeningitic  serum  is  not  refined  (Sophian 
reports  60  per  cent,  of  his  cases  as  developing  the  condition),  and  while 
the  symptoms  are  distressing,  they  are  seldom  alarming,  and  fatal 
anaphylaxis  is  extremely  rare.  Occasionally  the  onset  of  serum  sick- 
ness may  be  confusing,  but  if  the  meningeal  condition  has  been  respond- 
ing as  well  as  could  be  expected,  it  is  wise  to  let  the  patient  alone,  rather 
than  to  make  additional  punctures  and  cause  further  depression.  Local 
sedatives,  laxatives,  atropin,  sedatives,  and,  at  times,  morphin,  are 
indicated. 

Results  of  the  Serum  Treatment  on  Meningococcus  Meningitis. — 
(a)  Upon  the  Course  of  the  Disease. — In  the  majority  of  cases  the  sub- 
dural  injection  of  a  potent  antimeningitic  serum  is  followed  by  some 
immediate  improvement  in  the  local  suppurative  meningitis  and  general 
sepsis,  for  the  temperature  usually  drops,  the  mental  condition  improves, 
and  delirium  is  diminished,  although  the  Kernig  sign  may  persist,  partly 
as  the  result  of  meningeal  irritation  and  partly  on  account  of  fear. 
Hydrocephalus  is  generally  relieved,  as  indicated  by  lessening  of  the 
pressure  symptoms,  as,  for  example,  severe  headache,  vertigo,  and 
vomiting;  breathing  becomes  more  regular,  and  the  pulse  also  becomes 
slower  and  more  regular.  The  duration  of  the  illness  is  usually  shortened. 


800 


PASSIVE    IMMUNIZATION — SERUM   THERAPY 


According  to  Holt,  in  the  New  York  epidemic  of  1904-05,  antedating  the 
use  of  serum,  among  350  cases  that  recovered  the  duration  in  3  per  cent, 
was  one  week  or  less,  and  in  50  per  cent,  five  weeks  or  longer.  Of  288 
cases  reported  by  Flexner  and  Jobling,  the  average  duration  of  active 
symptoms  in  those  receiving  serum  was  eleven  days.  Sophian,  in  an 
experience  of  several  hundred  cases,  reports  that  many  acute  cases  were 
relieved  in  five  or  six  days  and  discharged  as  cured  in  two  weeks. 

(6)  Upon  Complications. — Next  to  its  influence  upon  mortality,  the 
good  effects  of  antimeningitic  serum  are  apparent  in  that  it  lessens  the 
incidence  and  severity  of  the  terrible  complications  of  this  disease.  The 
most  severe  and  permanent  sequels  are  those  resulting  from  affections 
of  the  internal  ear  and  the  essential  structures  of  vision.  A  conservative 
estimate  of  the  incidence  of  the  former  among  cases  not  receiving  serum 
treatment  is  12  per  cent.  (Goffert),  whereas  Flexner's1  analysis  of  1300 
cases  treated  with  serum  shows  that  deafness  occurred  in  but  3.5  per 
cent,  of  cases.  At  least  from  12  to  24  per  cent,  of  cases  not  receiving 
serum  treatment  will  develop  more  or  less  serious  eye  complications, 
whereas  Flexner's  report  shows  that  impairment  of  vision  among  serum- 
treated  cases  occurred  in  about  0.9  per  cent,  of  cases.  The  latter  report 
shows  the  occurrence  of  arthritis  in  but  0.9  per  cent,  of  cases,  whereas 
among  cases  untreated  with  serum  this  is  a  frequent  complication. 
Whereas  chronic  meningitis  is  relatively  common  among  cases  treated 
without  serum,  it  is  uncommon  among  those  treated  with  serum. 

(c)  Upon  Mortality. — The  gross  mortality  among  cases  treated 
without  serum  varies  from  70  to  90  per  cent.;  among  serum-treated 
cases  the  mortality  is  about  30  per  cent.  For  example,  of  1294  cases 
treated  with  serum  prepared  in  the  Rockefeller  Institute,  the  general 
mortality  was  30.9  per  cent. 

The  importance  of  early  diagnosis  and  prompt  institution  of  serum 
treatment  is  shown  in  the  following  table : 

TABLE  30.— MORTALITY  ACCORDING  TO  THE  PERIOD  OF  INJECTION 

OF  SERUM 


MORTALITY,  PER  CENT. 

TIME  OF  INJECTION 

Flexner, 

Dopter, 

Netter  and 

Sophian, 

1294  Cases 

402  Cases 

Debre,  99  Cases 

161  Cases 

First  to  third  day  

18.1 

8.2 

20.9 

9.0 

Fourth  to  seventh  day  .... 

27.2 

14.4 

33.3 

14.9 

Later  than  seventh  day  .  .  . 

36.5 

24.1 

26.0 

22.6 

Average  mortality  

30.8 

16.44 

28.0 

15.5 

1  Jour.  Exper.  Med.,  1913,  xvii,  553. 


THE    SERUM   TREATMENT    OF   MENINGOCOCCUS   MENINGITIS        801 


The  influence  of  age  upon  mortality  was  early  pointed  out  by  Flexner. 
The  very  high  mortality  in  infants  and  in  old  persons  is  due  to  their 
lowered  vitality  and  enfeebled  resistance.  An  additional  factor  in 
young  children  is  their  greater  tendency  to  develop  extreme  hydro- 
cephalus  and  convulsions. 

TABLE  31.— MORTALITY  ACCORDING  TO  AGE 


REPORT 

rED  BY 

Flexner 

Dopter 

Netter 

Sophian 

Under  one  year  
One  to  two  years  
Two  to  five  years  

49.6 
31.0 

28.4 

48.6 
20.1 
9.3 

50.0 
0.0 
16.6 

50.0 
21.2 

17.5 

Five  to  ten  years  

15.1 

8.5 

12.5 

9.0 

Ten  to  twenty  years  

29.4 

10.2 

0.0 

18.0 

Over  twenty  years  or  age 
not  given                        .  . 

38.2 

14.1 

0.0 

32.0 

The  statistics  show  indubitably  that  the  mortality  of  epidemic 
meningitis  can  be  greatly  reduced  by  the  administration  of  serum  treat- 
ment. While  the  ordinary  type  of  epidemic  meningitis  responds  best 
to  the  specific  treatment,  the  fulminant  cases  may  also  receive  some 
of  the  beneficial  influence  of  the  serum.  To  quote  from  what  Flexner 
wrote  in  1909,  and  repeated  in  1913:  "In  view  of  the  various  considera- 
tions presented,  the  conclusions  may  be  drawn  that  the  antimeningitis 
serum,  when  used  by  the  subdural  method  of  injection,  in  suitable  doses 
and  at  proper  intervals,  is  capable  of  reducing  the  period  of  illness;  of 
preventing  in  large  measure  the  chronic  lesions  and  types  of  the  infection, 
of  bringing  about  complete  restoration  of  health,  thus  lessening  the 
serious,  deforming,  and  permanent  consequences  of  meningitis;  and  of 
greatly  diminishing  the  fatalities  due  to  the  disease." 

Prophylactic  Immunization  in  Meningococcus  Meningitis. — In  Chap- 
ter XXIX  mention  has  been  made  of  the  probable  value  of  active  im- 
munization against  epidemic  meningitis  by  the  subcutaneous  injections 
of  three  doses  of  a  polyvalent  meningococcus  vaccine  at  intervals  of  a 
week.  Sophian  and  others  have  shown  experimentally  that  opsonins, 
bacteriolysins,  agglutinins,  and  other  antibodies  are  produced,  and 
while  meningococcus  meningitis  is  ordinarily  but  mildly  infectious  (about 
5  per  cent,  of  secondary  cases  in  homes),  the  method  is  practically 
devoid  of  danger  and  worthy  of  trial,  especially  for  physicians,  nurses, 
and  members  of  a  household  who  are  exposed  to  the  infection  over  a 
period  of  many  weeks. 
51 


802  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

Passive  immunization  by  means  of  the  subcutaneous  injection  of 
antimeningitic  serum  was  advised  by  Jochmann  in  1906,  but  has  not 
come  into  general  use.  The  immunity  resulting  from  the  injection  of 
from  10  to  20  c.c.  of  serum  is  only  temporary,  and  probably  lasts  about 
a  month.  Another  drawback  is  the  resulting  serum  sensitization,  which 
renders  subsequent  injections  of  serum  more  likely  to  be  followed  by 
serum  sickness.  In  the  presence  of  an  active  epidemic,  such  as  that 
which  occurred  in  Texas  during  1912,  immediate  passive  immunization 
of  physicians,  nurses,  and  attendants  by  the  subcutaneous  injection  of 
15  c.c.  of  serum,  followed  by  active  immunization  with  three  doses  of 
vaccine  (500,  1000,  and  1000  millions)  at  intervals  of  a  week,  may  be 
advisable.  While  there  are  no  available  statistics  to  prove  the  value  of 
this  procedure,  it  is,  at  least,  a  rational  one,  and  since  there  is  danger  of 
contracting  the  disease,  especially  after  prolonged  contact,  physicians 
should  practise  immunization  during  epidemics  of  this  dreaded  disease. 
In  mixed  passive  and  active  immunization  the  serum  probably  affords 
immediate  protection,  and  tides  over  any  temporary  negative  phase  or 
period  of  lowered  resistance  following  the  injections  of  vaccine. 


THE  SERUM  TREATMENT  OF  INFLUENZAL  MENINGITIS 
Since  lumbar  puncture  as  an  aid  to  the  diagnosis  of  meningitis  is 
coming  into  more  general  use,  the  important  fact  has  been  revealed  that 
the  influenza  bacillus  is  not  an  infrequent  cause  of  severe,  and  usually 
fatal,  seropurulent  cerebrospinal  meningitis.  In  1911  Wollstein1  col- 
lected 58  cases  of  this  infection,  all  but  6  ending  fatally,  and  as  the  bac- 
terial diagnosis  of  meningitis  is  becoming  more  widely  known  and  more 
commonly  employed,  the  number  of  reported  cases  is  increasing  rapidly. 
The  mortality  of  90  per  cent.,  which  is  exceeded  only  by  the  tuberculous 
and  pneumococcus  infections  of  the  meninges,  and  the  encouraging  re- 
sults following  the  use  of  a  specific  anti-influenzal  serum  in  experimental 
infections  in  monkeys,  render  this  subject  one  of  great  importance  from 
the  standpoint  of  serum  therapy. 

Influenzal  Meningitis. — Like  the  acute  meningeal  infections  in 
general,  influenzal  meningitis  is  more  prevalent  among  children  than 
among  adults. 

Infection  of  the  meninges  is  probably  always  secondary  to  infection 
of  the  respiratory  tract  with  virulent  influenza  bacilli,  the  route  of  in- 
fection being  chiefly  through  the  blood-current.     Direct  infection  from 
1  Jour.  Exper.  Med.,  1911,  xiv,  73;  Amer.  Jour.  Dis.  Child.,  1911,  i,  42. 


THE    SERUM   TREATMENT    OF    INFLUENZAL   MENINGITIS  803 

the  nose  cannot  be  excluded,  and  should  be  considered  a  possibility. 
However,  all  or  nearly  all  cases  of  spontaneous  influenzal  meningitis  in 
human  beings  are  the  result  of  influenzal  bacteremia,  since  the  bacilli 
have  been  cultivated  in  large  numbers  from  the  heart's  blood  before  and 
after  death.  The  same  is  true  of  experimental  influenzal  meningitis  in 
the  monkey. 

According  to  Flexner,1  the  cerebrospinal  fluid  removed  by  lumbar 
puncture  from  human  patients  is  always  turbid,  and  deposits  a  yellowish 
or  whitish  sediment  on  standing.  "As  the  disease  advances,  the  fluid 
becomes  more  heavily  charged  with  pus-cells,  until  toward  the  end,  and 
as  late  as  the  seventh  day  of  illness,  the  puncture  may  yield  merely  a 
viscid  mass  of  purulent  matter.  The  number  of  influenza  bacilli  pre- 
sent in  the  fluid  is  usually  large,  and  the  bacilli  lie  chiefly  extracellular, 
among  the  pus-cells,  although  a  variable  but  small  number  is  usually 
found  ingested  by  the  leukocytes.  In  morphology  the  bacilli  vary 
somewhat,  and  in  this  respect  the  observer  may  readily  be  deceived  as 
to  the  nature  of  the  bacteria  present.  While  some  of  the  fluids  contain 
the  typical,  minute  rods,  others  show  quite  irregular  and  knobbed  or 
even  filamentous  bacteria  that  have  little  resemblance  to  the  influenza 
bacillus  as  seen  in  recent  cultivations.  These  bizarre  or  involution 
forms,  however,  are  met  in  old  and  exhausted  cultures;  and  when  they 
are  recultivated  on  a  suitable  hemoglobin  medium,  they  yield  the  typical 
minute  rods." 

The  cerebrospinal  fluid  removed  from  monkeys  inoculated  by  sub- 
dural  injection  with  virulent  cultures  of  the  influenzal  bacillus  resembles 
in  all  essential  particulars  the  fluid  removed  from  patients  with  spon- 
taneous infections. 

The  bacteriologic  diagnosis  can  usually  be  made  by  microscopic 
examination  of  stained  smears  of  the  fluid,  but  whenever  possible,  the 
diagnosis  should  be  confirmed  by  cultural  methods. 

Anti-influenza  Serum. — After  having  satisfactorily  demonstrated 
experimental  influenza  meningitis  in  the  monkey,  Flexner  and  Wollstein 
prepared  an  immune  serum  and  showed  that  the  experimental  infection 
could  be  controlled  and  cured  by  injecting  the  serum  directly  into  the 
seat  of  disease  by  intraspinal  inoculation.  The  immune  serum  was 
prepared  by  the  ordinary  methods,  first  a  goat  and  then  a  horse  being 
injected  with  non- virulent  and  finally  with  virulent  bacilli,  covering  a 
period  of  many  months,  until  their  serums  showed  the  presence  of 
agglutinins  and  bacteriotropins.  The  serum  lacked  bacteriolytic  prop- 
1  Jour.  Amer.  Med.  Assoc.,  1913,  Ixi,  1872. 


804  PASSIVE   IMMUNIZATION — SERUM   THERAPY 

erties,  and  did  not  give  rise  to  complement  fixation  in  dilutions  greater 
than  1  : 100. 

Following  the  administration  of  serum,  the  cerebrospinal  fluid  tends 
to  clear  up;  the  bacilli  become  fewer  in  number  and  are  mostly  ingested 
by  the  phagocytes;  the  pus-cells  become  less  numerous,  and  while  the 
bacilli  may  persist  in  the  fluid  for  a  longer  period,  they  ultimately  dis- 
appear. 

Administration  of  Anti-influenza  Serum. — The  Rockefeller  Institute 
has  distributed  serum  throughout  different  parts  of  the  country,  and  is 
prepared  to  furnish  it  to  physicians  upon  request.  Physicians,  and 
especially  pediatrists,  should  resort  to  lumbar  puncture  early  in  all  sus- 
pected cases  of  meningitis,  for  only  in  this  manner  may  influenzal  menin- 
gitis be  detected  early  enough  to  derive  any  possible  benefit  from  serum  treat- 
ment. The  serum  should  be  injected  directly  into\  the  spinal  canal  by  the 
gravity  or  syringe  method  in  exactly  the  same  manner  and  with  the  same 
precautions  as  are  observed  in  administering  serum  in  the  treatment  of 
epidemic  meningitis.  Since  the  disease  is  usually  accompanied  by  a 
bacteremia,  it  is  well  to  inject  serum  intravenously,  although  serum  in- 
jected intraspinally  soon  finds  its  way  into  the  blood-stream.  All  data, 
including  the  clinical  history  and  the  records  of  bacteriologic  examina- 
tions of  cerebrospinal  fluid  and  blood,  the  amounts  of  serum  injected, 
and  the  results  obtained,  should  be  sent  to  the  Director  of  the  Rocke- 
feller Institute. 


THE  SERUM  TREATMENT  OF  PNEUMOCOCCUS  MENINGITIS 
Meningitis  is  caused  more  frequently  by  the  pneumococcus  than  by 
the  influenza  bacillus.     Its  mortality  is  certainly  no  less  than  in  influ- 
enzal meningitis. 

The  few  instances  in  which  antipneumococcic  serum  has  been  em- 
ployed have  not  yielded  results  that  inspire  confidence  in  its  employ- 
ment alone.  As  will  be  emphasized  later,  in  considering  the  serum 
treatment  of  pneumonia,  an  antipneumococcus  serum  is  at  best  active  only 
against  the  homologous  organism  or  organisms,  the  types  of  which  have  been 
employed  in  its  preparation.  Even  when  the  homologous  serum  is  used 
in  treating  experimental  pneumococcus  meningitis  in  monkeys,  the 
fatal  termination  may  be  delayed,  but  is  not  prevented.  For  this 
reason  the  outlook  for  its  successful  employment  alone  in  human  in- 
fections is  not  encouraging.  Recent  investigations  of  Lamar1  have 
1  Jour.  Exper.  Med.,  1911,  i;  ibid.,  380;  xiv,  256;  1912,  xvi,  581. 


THE   SERUM   TREATMENT   OF   PNEUMOCOCCUS   MENINGITIS          805 

shown,  however,  that  mixtures  of  homologous  antipneumococcus  serum, 
sodium  oleate,  and  boric  acid  exert  a  marked  and  decided  curative  in- 
fluence upon  a  virulent  experimental  meningitis,  and  while  this  method 
has  not  thus  far  been  generally  applied  in  the  treatment  of  the  disease 
in  humans,  it  is  deserving  of  trial  and  offers  considerable  encouragement 
for  an  otherwise  highly  fatal  infection. 

Pneumococcus  Meningitis. — This  infection  is  usually  secondary, 
and  follows  on  pneumonia  or  on  inflammations  of  serous  membranes  by 
indirect  transmission  by  the  blood  or  by  direct  infection  from  the  naso- 
pharynx, mastoid  cells,  frontal,  sphenoid  and  ethmoid  sinuses,  and 
internal  ear. 

The  diagnosis  is  usually  made  as  the  result  of  microscopic  examination 
of  stained  smears  of  cerebrospinal  fluid  removed  by  lumbar  puncture. 
Large  numbers  of  polynuclear  leukocytes  with  intracellular  and  extra- 
cellular Gram-positive  diplococci,  occurring  in  pairs  or  in  short  chains, 
usually  indicate  a  pneumococcus  infection.  Whenever  possible,  the 
diagnosis  should  be  confirmed  by  making  cultures  of  the  fluid  on  dextrose 
blood  agar,  and  by  injecting  portions  intraperitoneally  and  subcutane- 
ously  in  mice. 

Pneumococcus  infections  of  the  cerebral  meninges  have  been  found 
experimentally  to  be  more  refractory  to  treatment  than  infections  of  the 
spinal  meninges,  hence  human  infections  following  injuries  to  the  head,  or 
occurring  as  the  result  of  direct  extension  from  neighboring  sinuses,  are 
likely  to  be  more  refractory  than  indirect  infections  by  way  of  the  blood. 

Serum  Treatment. — Numerous  investigations  by  Conradi,1  Korschun 
and  Morgenroth,2  Levaditi,3  and  Noguchi4  have  shown  that  substances 
may  be  obtained  directly  from  tissue-cells  and  leukocytes  or  after  auto- 
lysis  which  are  bactericidal  and  hemolytic,  and,  as  shown  by  Noguchi, 
are  largely  in  the  nature  of  higher  saturated  fatty  acids  or  their  alkaline 
soaps.  (For  an  account  of  their  similarity  to  complements  see  Chapter 
XVIII.)  As  shown  by  Klotz,5  soaps  occur  in  inflammatory  foci,  and  the 
origin  of  the  fatty  acids  and  soaps  is  readily  accounted  for  since  Achaline6 
has  shown  the  presence  of  lipase  in  such  foci.  With  the  death  of  leuko- 
cytes in  an  inflammatory  focus,  brought  about  by  a  bacterial  poison, 
leukocidins,  or  lack  of  nutriment,  disintegration  occurs,  and  by  auto- 

1  Beit.  z.  chem.  Phys.  u.  Path.,  1902,  i,  193. 

2  Berl.  klin.  Wochenschr.,  1902,  xxxix,  870. 

3  Ann.  de  1'Inst.  Pasteur,  1903,  xvii,  187. 

4  Biochem.  Zeitschr.,  1907,  vi,  327.  6  Jour.  Exper.  Med.,  1905,  vii,  633. 
6  Compt.  rend.  Soc.  de  biol.,  1899,  11,  568. 


806  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

lysis  and  lipolysis  fatty  acids  and  soaps  are  produced  that  in  themselves 
seem  to  exert  a  destructive  action  upon  the  infecting  bacteria. 

With  these  considerations  in  mind,  Lamar  investigated  the  influence 
of  soaps  upon  pneumococci.  Solutions  of  0.5  to  1  per  cent,  of  sodium 
oleate  were  found  rapidly  to  kill  pneumococci;  much  weaker  solutions, 
as,  e.  g.,  0.1  per  cent.,  or  even  1  part  of  soap  in  10,000  parts  of  water, 
were  found  to  lessen  their  virulence,  and,  what  is  more  important  and 
significant,  to  render  the  organisms  peculiarly  susceptible  to  lysis  by  a 
homologous  antipneumococcus  serum. 

A  serious  drawback  to  the  application  of  these  discoveries  was  that 
protein  constituents  of  serum  and  exudates  were  found  to  inhibit  the 
bacteriolytic  and  hemolytic  action  of  unsaturated  fatty  acid  soaps,  as 
was  shown  by  Noguchi l  and  then  by  von  Liebermann.2  The  latter  and 
von  Fenyvessy3  later  found  that  this  inhibition  can  be  prevented  in  the 
test-tube  and  also  in  the  animal  body  by  adding  a  minute  quantity  of 
boric  add,  which  prevents  the  union  of  soap  and  protein  matter  when  the 
latter  is  not  too  greatly  in  excess. 

The  experiments  of  Lamar  with  mixtures  of  homologous  antipneu- 
mococcus serum,  sodium  oleate,  and  boric  acid  in  the  treatment  of  pneu- 
mococcus  meningitis  in  the  monkey  have  yielded  excellent  results, 
especially  when  used  early  in  the  infection.  These  experiments  have 
proved  that  sodium  oleate  lowers  the  virulence  of  pneumococci  and 
renders  them  peculiarly  and  highly  susceptible  to  solution  by  bacter- 
iolysins  present  in  the  serum,  and  that  boric  acid  largely  prevents  the 
inhibitory  action  of  protein  constituents  upon  this  sensitizing  action  of 
sodium  oleate. 

Practical  Applications. — One  drawback  to  the  use  of  this  method  in 
the  treatment  of  human  infections  is  the  necessity  of  using  an  anti- 
pneumococcus serum  corresponding  to  the  organism  causing  the  infec- 
tion. In  the  Rockefeller  Hospital  a  method  has  been  worked  out  where- 
by the  type  of  infection  is  quickly  determined  by  agglutination  reactions. 
Fortunately,  the  number  of  types  of  pneumococci  are  relatively  few, 
and  it  is  to  be  hoped  that  an  efficient  polyvalent  serum  will  soon  be  made 
available  by  the  Rockefeller  Institute.  Until  this  desideratum  is 
attained,  the  commercial  serums  at  present  on  the  market  may  be  used, 
with  the  addition  of  sodium  oleate  and  boric  acid. 

It  is  highly  desirable  that  the  treatment  be  administered  as  early  as 
possible,  when  the  exudate  is  largely  serous  or  at  most  seropurulent. 

Loc.  cit.  2  Biochem.  Zeitschr.,  1907,  iv,  25 

3  Zeitschr.  f.  Immunitatsforsch.,  orig.,  1909,  ii,  436. 


SERUM  TREATMENT   OF   LOCALIZED   PNEUMOCOCCUS   INFECTIONS   807 

The  mixture  should  be  injected  into  the  spinal  canal  after  the  with- 
drawal of  the  fluid  by  the  gravity  or  syringe  method  and  under  blood- 
pressure  control,  as  previously  described.  The  amount  injected  and  the 
number  of  injections  depend  upon  the  clinical  condition  of  the  patient, 
and  in  general  may  be  administered  in  the  same  way  as  is  antimenin- 
gitic  serum. 

The  initial  dose  may  be  20  c.c.,  and  is  prepared  as  follows: 

Antipneumococcus  serum  (sterile) ;  .  4  c.c. 

5  per  cent,  aqueous  solution  of  boric  acid  (sterile) 15  c.c. 

2  per  cent,  aqueous  solution  of  sodium  oleate  (Kahlbaum's 
or  Merck's)  (sterile) 1  c.c. 


THE    SERUM  TREATMENT    OF    OTHER    LOCALIZED    PNEUMOCOCCUS 

INFECTIONS 

Lamar  has  also  suggested  that  mixtures  of  antipneumococcus  serum, 
sodium  oleate,  and  boric  acid  may  be  used  in  the  treatment  of  pneumo- 
coccus  pleuritis,  peritonitis,  or  other  localized  infections,  such  as  arthritis 
and  sinusitis,  where  the  exudate  may  be  removed  and  the  serum  be 
brought  into  contact  with  the  infected  tissues. 

THE  SERUM  TREATMENT  OF  PNEUMONIA 

Acute  lobar  pneumonia,  with  its  clear-cut  clinical  course,  unsatis- 
factory and  difficult  treatment,  uncertain  prognosis,  and  high  mortality, 
was  one  of  the  diseases  in  which  the  earliest  efforts  were  directed  toward 
discovering  a  specific  serum  therapy.  Since  the  pioneer  work  of  the 
Klemperers  in  1891,  numerous  investigators  have  prepared  serums  that 
have  yielded  either  indifferent  results  or  proved  beneficial  in  but  a  limited 
number  of  cases,  so  that  there  has  been  no  well-established  form  of 
specific  therapy. 

Recent  investigations  by  Neuf eld  and  Handel 1  in  Germany,  and  by 
Dochez,2  Cole,3  and  Gillespie4  in  the  Rockefeller  Institute,  have  dis- 
closed several  reasons  for  the  failure  of  serum  therapy  in  pneumonia, 
and  have  emphasized  the  importance  of  the  following  factors : 

1.  The  serum  should  correspond  to  the  type  of  pneumococcus  causing 
the  infection. 

1  Zeitschr.  f.  Immunitatsforsch.  1909,  iii,  159;   Arb.  a.  d.  k.  Gesundheitsamte, 
1910,  xxxiv,  169;   ibid.,  1910,  xxxiv,  293;   Berl.  klin.  Wochenschr.,  1912,  xlix,  680. 

2  Jour.  Exper.  Med.,  1912,  xvi,  665,  680,  and  693;    Jour.  Amer.  Med.  Assoc., 
1913,  Ixi,  727. 

3  Jour.  Exper.  Med.,  1912,  xvi,  644;   Arch.  Int.  Med.,  1914,  xiv,  56. 

4  Jour.  Amer.  Med.  Assoc.,  1913,  Ixi,  727;   Jour.  Exper.  Med.,  1914,  xix,  28. 


808  PASSIVE   IMMUNIZATION — SERUM  THERAPY 

2.  The  serum  must  be  administered  in  large  doses,  and  preferably 
intravenously. 

3.  To  be  most  effective  the  treatment  should  be  given  as  early  as 
possible. 

The  investigators  in  the  Rockefeller  Institute  have  divided  the 
pneumococci  causing  lobar  pneumonia  into  four  main  groups,  and  have 
worked  out  a  method  for  the  rapid  identification  and  classification  of  the 
particular  pneumococcus  that  is  the  etiologic  factor  in  a  given  infection, 
so  that  with  the  proper  administration  of  the  corresponding  immune 
serum  very  encouraging  results  have  been  obtained  in  the  serum  treat- 
ment of  pneumonia.  These  researches  are  of  importance  not  only  in 
this  connection,  but  also  from  the  fact  that  they  may  have  disclosed  the 
reasons  for  failure  in  the  treatment  of  streptococcus  and  other  infec- 
tions, and  that  similar  studies  in  these  conditions  may  insure  for  serum 
therapy  a  definite  and  valuable  role  in  the  treatment  of  disease. 

The  Nature  of  Lobar  Pneumonia. — The  frequency  with  which  the 
Diplococcus  pneumonia  is  found  in  the  local  lesion  and  in  severe  cases  in 
the  blood-stream  of  pneumonia  patients,  and  the  more  recent  experi- 
mental studies  of  Wadsworth,1  Meltzer,2  Wollstein  and  Meltzer,3  Win- 
ternitz,  Kline  and  Hirschfelder,4  leave  little  doubt  regarding  the  etio- 
logic relationship  of  this  microorganism  to  lobar  pneumonia.  Much  still 
remains  to  be  learned,  however,  regarding  the  method  of  infection  and 
the  nature  of  the  resulting  disease.  While  pneumococci  are  to  be  found 
living  in  the  upper  air-passages  as  harmless  parasites,  it  is  probable  that 
those  causing  infection  differ  inherently  as  regards  adaptation  or  viru- 
lence for  man.  In  addition,  it  is  likely  that  general  resistance  is  low- 
ered in  some  more  or  less  peculiar  manner,  and  experimental  studies 
in  animals,  as  well  as  the  course  of  the  disease  in  man,  suggest  most 
strongly  that  local  changes  in  the  respiratory  tract  may  precede  the 
infection,  so  that  a  combination  of  factors,  such  as  the  virulence  of  the 
organisms  and  the  diminished  general  and  local  resistance,  plays  a  part 
in  the  production  of  lobar  pneumonia. 

In  whatever  manner  produced,  the  disease  is  finally  to  be  regarded 
as  a  general  infection,  with  localization  of  the  process  in  the  lung.  While 
pneumococci  may  be  found  in  the  blood  of  the  most  severe  cases,  the 
general  symptoms  are  apparently  due  to  intoxication  with  a  poison  or 
toxin  derived  primarily  from  the  pneumococci,  and  secondarily  from  the 

1  Amer.  Jour.  Med.  Sci.,  1904,  cxxvii,  851.  2  Jour.  Exper.  Med.,  1912,  xv,  133. 

3  Jour.  Exper.  Med.,  1913,  xvii,  353,  RWR;   ibid.,  1913,  xviii,  548. 

4  Jour.  Exper.  Med.,  1912,  xvii,  657;  ibid.,  1913,  xviii,  50. 


SERUM   TREATMENT    OF   LOCALIZED    PNEUMOCOCCUS    INFECTIONS    809 

exudate  in  the  local  lesions.  The  studies  of  Rowntree,1  Medigreceanu,2 
and  Peabody,3  showing  chlorin  retention;  of  Peabody,4  showing  pro- 
gressive loss  in  the  oxygen-combining  power  of  the  hemoglobin,  due  to 
the  formation  of  methemoglobin;  of  Medigreceanu,5  showing  a  deficiency 
of  oxydase  or  lessened  power  of  the  tissues  to  carry  on  proper  oxidation, 
and  of  Neufeld  and  Bold,6  Rosenow,7  Cole,8  Jobling  and  Strouse,9 
indicating  the  presence  of  endotoxins  within  the  pneumococci — all  these 
support  the  view  that  in  pneumonia  there  is  well-marked  intoxication, 
and  this,  in  addition  to  the  effects  of  the  local  pulmonary  consolidation 
on  the  heart,  respiration,  and  nervous  system,  constitute  the  main  fea- 
tures of  the  infection. 

.  Regarding  the  mechanism  of  recovery  from  pneumonia,  there  is 
little  definite  information.  The  recent  studies  of  Neufeld,  Dochez,  and 
Clough  indicate  that  antibodies  are  produced  at  or  about  the  time  of  the 
crisis,  and  that  these  are  probably  responsible  for  the  destruction  of  the 
bacteria  in  the  circulating  blood,  and,  to  a  greater  extent,  in  the  local 
lesion.  In  the  resolution  of  the  local  lesion  it  is  probable  that  ferments 
play  an  important  part.  That  resolution  does  not  occur  earlier  may  be 
due  to  the  overbalancing  of  the  leukocytic  ferments  by  the  antiferments 
of  the  serum,  and  the  lytic  ferments  become  active  only  when  they  reach 
a  point  of  excess  over  the  antiferments,  causing  a  solution  of  the  fibrin, 
relieving  tension,  and  affording  an  outlet  for  the  exudate.  According 
to  Vaughan,  the  pneumococci  may  be  considered  as  furnishing  a  ferment 
that  brings  about  the  production  of  a  specific  antiferment,  capable  of 
reacting  upon  its  substratum,  the  ferment  and  the  new  bacterial  tissue, 
and  causing  its  destruction  by  a  process  of  solution.  Pneumococci  in 
the  resolving  lesion  are  probably  destroyed  by  leukocidins  released 
through  disintegration  of  leukocytes,  by  fatty  acids,  and  probably  by 
antibacterial  substances  in  the  blood. 

While  it  is  true  that  immunity  does  not  usually  follow  an  attack  of 
pneumonia,  and,  indeed,  the  patient  is  apparently  hypersusceptible,  it 
has  been  found  experimentally  that  the  antibodies  are  highly  specific 
for  the  particular  organism  causing  an  infection.  Reinfection  is,  there- 
fore, possible  with  an  organism  belonging  to  another  group,  and  lia- 


1  Bull.  Johns  Hopkins  Hosp.,  1908,  xix,  367.       2  Jour.  Exper.  Med.,  1911,  xiv,  289. 
3  Jour.  Exper.  Med.,  1913,  xvii,  71.  4  Jour.  Exper.  Med.,  1913,  xviii,  7. 

6  Jour.  Exper.  Med.,  1914,  xix,  309. 

6  Berl.  klin.  Wochenschr.,  1911,  xlviii,  1069. 

7  Jour.  Infec.  Dis.,  1911,  ix,  190.  8  Jour.  Exper.  Med.,  1912,  xvi,  644. 
9  Jour.  Exper.  Med.,  1913,  xviii,  597. 


810  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

bility  to  reinfection  may  be  increased  because  of  lowered  local  and  gen- 
eral resistance  due  to  the  previous  attack. 

Antipneumococcus  Serum. — The  indications  of  specific  serum 
therapy,  are,  therefore,  mainly  twofold:  first,  to  destroy  any  pneumo- 
cocci  present  in  the  blood  and  in  the  local  lesion;  or  if  the  latter  is  im- 
possible because  of  mechanical  obstacles  that  interfere  with  the  circula- 
tion and  prevent  access  of  the  antibodies  to  the  cocci,  to  at  least  prevent 
extension  of  the  lesion  by  preventing  the  multiplication  of  organisms  at 
its  margin;  second,  to  neutralize  the  toxins  produced  during  the  course 
of  the  disease. 

It  would,  of  course,  be  highly  desirable  to  have  at  our  command  a 
serum  that  would  cause  solution  of  the  local  exudate  and  bring  about  a 
crisis  and  a  cure.  It  is  hardly  reasonable  to  expect,  however,  that  a 
serum  can  be  produced  that  will  contain  digestants  for  fibrin  and  leuko- 
cytes. The  local  lesion  is  most  likely  to  be  harmful  because  of  the  toxic 
substances  that  emanate  from  it,  and  not  because  so  large  an  area  of 
lung  is  temporarily  incapacitated  and  the  heart  embarrassed.  A  serum 
that  will  prevent  general  bacteremia,  limit  the  extension  of  the  local 
lesion,  and  neutralize  the  toxins  while  nature  is  preparing  to  react  upon 
the  exudate  with  a  ferment,  is  probably  fulfilling  all  that  may  be  ex- 
pected of  a  specific  serum  therapy. 

Groups  of  Pneumococci. — Neufeld  and  Handel  have  shown  that  an  immune 
serum  produced  by  the  injection  of  a  given  variety  of  pneumococci  into  an  animal 
was  not  effective  against  all  forms  of  pneumococci.  In  the  Rockefeller  Hospital  a 
serum,  known  as  Serum  1,  prepared  by  immunizing  a  horse  with  a  culture  obtained 
from  Neufeld,  was  found  to  protect  against  only  about  one-half  the  types  of  pneu- 
mococci (Group  1)  studied.  By  immunizing  rabbits  to  each  of  the  types  that  were 
not  acted  upon  by  Serum  1,  and  testing  the  immune  serum  against  all  strains  by 
cross-agglutination  and  by  cross-protection  experiments,  it  was  found  that  a  number 
of  the  serums  possessed  the  same  properties,  thus  indicating  that  their  respective 
cultures  belonged  to  the  same  general  group  (Group  2).  By  immunizing  a  horse  with 
one  of  these,  Serum  2  was  produced.  In  Group  3  are  placed  all  the  organisms  of  the 
so-called  Pneumococcus  mucosus  type.  In  Group  4  are  included  all  the  varieties  of 
pneumococci  against  which  Serums  1  and  2  are  not  effective,  and  which,  from  their 
other  properties,  do  not  belong  in  Group  3.  Animals  may  readily  be  immunized  to 
any  member  of  this  Group  4,  and  the  serum  of  the  immunized  animal  is  protective 
against  the  strain  used  for  immunization,  but  in  no  instance  has  this  serum  been 
found  effective  against  any  other  member  of  this  group  or  against  the  organisms  of 
the  other  groups.  While  no  cultural  or  morphologic  differences  between  the  members 
of  Group  1,  2,  and  4  exist,  it  has  been  found  possible  to  group  them  by  the  aggluti- 
nation reaction  in  exactly  the  same  manner  as  by  protection  experiments.  Of 
24  strains  studied  in  the  Rockefeller  Hospital,  47  per  cent,  belonged  to  Group  1, 
18  per  cent,  to  Group  2,  13  per  cent,  to  Group  3,  and  22  per  cent,  to  Group  4. 

Determining  the  Type  of  Pneumococcus. — The  method  is  described  on  page  308. 

Preparation  of  Antipneumococcus  Serum. — Horses  are  immunized  with  dead 
and  then  with  virulent  living  cultures.  In  view  of  the  fact  that  the  serum  should 


SERUM   TREATMENT   OF   LOCALIZED   PNEUMOCOCCUS   MENINGITIS    811 

possess  some  antitoxic  value,  it  is  desirable  that  the  animals  be  immunized  also  with 
autolysates.  The  whole  process  may  be  conducted  after  the  method  described  for 
the  production  of  antimeningococcus  serum. 

In  the  Rockefeller  Institute  different  horses  are  immunized  with  strains  belong- 
ing to  Groups  1  and  2.  It  would  appear  possible  to  produce  a  potent  polyvalent 
serum,  and  this  is  very  much  to  be  desired,  especially  if  further  studies  continue  to 
show  that  65  per  cent,  of  infections  are  caused  by  organisms  belonging  to  these  two 
groups. 

Kolle,  by  immunizing  horses  with  cultures  secured  from  pneumonia  patients, 
produces  an  antipneumococcic  serum.  These  cultures  are  grown  in  broth  for  forty- 
eight  hours,  heated  to  60°  C.,  and  5  c.c.  injected  intravenously  into  a  horse.  The 
dose  is  increased  each  week  until  120  c.c.  are  given  at  one  time.  Then  5  c.c.  of  living 
culture  is  injected,  and  the  doses  increased  in  a  similar  manner  until  120  c.c.  are  given 
at  one  time.  About  six  months  are  consumed  in  the  process  of  immunization,  and 
two  weeks  after  the  last  injection  has  been  given  the  serum  is  tested. 

Concentration  of  Antipneumococcus  Serum. — In  view  of  the  large  doses  of 
serum  required  in  the  treatment  of  lobar  pneumonia  and  the  possible  injurious 
effects  of  the  large  amount  of  horse-serum  protein  injected,  Gay  and  Chickering1  and 
Chickering2  have  endeavored  to  concentrate  the  immune  serum  by  adding  rela- 
tively small  amounts  of  the  extract  of  pneumococcus  and  recovering  the  resulting 
precipitate.  These  precipitates  have  been  found  to  contain  practically  all  the  pro- 
tective antibodies  of  the  original  serum  and  relatively  small  amounts  of  protein  as 
compared  with  the  original  serum. 

Standardization  of  Antipneumococcus  Serum. — As  previously  mentioned,  there 
is  at  present  no  accurate  method  for  standardizing  an  antibacterial  serum.  It 
is  possible,  however,  to  obtain  some  measure  of  its  protective  and  curative  power  by 
employing  various  tests. 

1.  Protective  Value. — The  lethal  dose  of  a  living  pneumococcus  culture  for  mice 
is  determined,  and  from  10  to  100  times  this  amount  of  culture  is  mixed  with  decreas- 
ing doses  of  immune  serum  and  the  mixtures  injected  subcutaneously  or  intraperitone- 
ally  into  a  series  of  mice  in  order  to  determine  the  dose  of  serum  that  will  protect. 
Dochez  has  found  that  when  these  mixtures  are  injected  at  once  and  in  the  same  place, 
the  serum  will  obey  the  law  of  multiple  proportions  up  to  a  certain  limit. 

Merck's  antipneumococcus  serum  is  so  standardized  that  0.01  c.c.  injected 
subcutaneously  protects  a  mouse  inoculated  intraperitoneally  twenty-four  hours 
later  with  from  10  to  100  times  the  lethal  dose  of  a  living  virulent  culture.  This  is 
known  as  a  normal  serum,  1  c.c.  containing  an  immunity  unit  (I.  U.).  The  serum  is 
marketed  in  vials  containing  from  20  to  40  c.c.  Kolle  regards  as  satisfactory  a  serum 
that,  in  doses  of  0.001  c.c.  and  less,  will  protect  mice. 

2.  Bacteriotropic  Value. — Neufeld  lays  considerable  stress  upon  this  point.    The 
technic  of  this  titration  has  been  described  elsewhere. 

3.  Complement- fixation  and   Agglutination    Tests. — While   these   tests   are   fre- 
quently sharply  cut,  and  while  they  serve  as  a  measure  for  record  in  the  laboratory, 
they  do  not  necessarily  indicate  the  therapeutic  value  of  the  serum. 

Action  of  Antipneumococcus  Serum. — The  curative  and  protective 
value  of  this  serum  depend  mainly  upon  bacteriolysins,  bacteriotropins, 
and  antitoxins.  The  first  are  readily  demonstrated  in  protection  ex- 
periments and  also  in  pneumonic  patients  when  pneumococci  in  the 
blood-stream  are  destroyed.  Bacteriotropins  may  likewise  be  demon- 
strated experimentally  and  in  the  blood  of  patients  if  the  corresponding 

1  Jour.  Exper.  Med.,  1915,  xxi,  389.  2  Jour.  Exper.  Med.,  1915,  xxii,  248. 


812  PASSIVE    IMMUNIZATION— SERUM    THERAPY 

organism  is  used  in  the  tests  (Neufeld,  Strouse).  The  antitoxic  prop- 
erties are  shown  clinically  and  also  in  vitro  by  neutralization  of  the  hemo- 
toxic  poison  obtained  by  dissolving  pneumococci  in  bile.  As  shown 
recently  by  Bull,1  the  agglutinins  in  the  serum  may  play  a  very  active 
role  by  producing  an  agglutination  of  the  cocci  in  the  blood-stream 
followed  by  phagocjrtosis. 

Administration  of  Antipneumococcus  Serum. — To  obtain  the  best 
results  the  serum  should  be  given  as  early  as  possible  and  intravenously. 
In  young  children,  when  the  giving  of  an  intravenous  injection  is  quite 
difficult  or  impossible,  the  muscles  of  the  buttocks  should  be  substituted. 
Certainly  small  doses  given  subcutaneously  are  almost  devoid  of  effect. 
The  procedure  in  use  in  the  Rockefeller  Institute  consists  in  injecting 
0.5  c.c.  of  serum  subcutaneously  to  discover  if  hypersensitiveness  exists 
and  to  produce  anti-anaphylaxis.  As  soon  as  the  type  of  organism  has 
been  determined  (see  page  308),  from  50  to  100  c.c.  of  the  serum,  diluted 
one-half  with  salt  solution,  are  injected  intravenously.  The  condition 
of  the  patient  serves  as  a  guide  in  the  later  treatment.  Usually  the 
serum  is  not  administered  oftener  than  once  every  twelve  hours. 

It  has  been  shown  experimentally  that,  in  the  presence  of  a  maximum 
degree  of  infection,  no  amount  of  serum,  however  large,  is  effective. 
This  suggests  that  the  body  must  furnish  a  second  substance  to  act  with 
the  antibodies  in  the  serum,  and  indicates  the  early  administration  of 
serum  before  the  infection  has  reached  too  extreme  a  grade.  I  would 
also  suggest  that  the  body  may  be  deficient  in  bacteriolytic  complements, 
and  that  the  effect  of  a  serum  may  be  enhanced  by  adding  fresh  sterile 
guinea-pig  serum — say  5  c.c.  to  each  100  c.c.  of  immune  serum — just 
prior  to  administration. 

Results  in  the  Serum  Treatment  of  Pneumonia. — It  is  hardly  neces- 
sary to  review  the  numerous  reports  that  have  been  made  in  past  years, 
because  in  most  instances  the  serum  was  administered  subcutaneously 
and  in  too  small  doses  to  be  of  value,  even  granting  that  it  contained 
antibodies  for  the  particular  infection.  Of  23  patients,  all  seriously  ill, 
treated  in  the  Rockefeller  Institute  during  the  past  year,  the  results 
were  as  follows:  Of  15  cases  due  to  pneumococcus  1,  all  recovered  but 
one — a  mortality  of  6.6.  per  cent.,  as  compared  with  the  mortality  of 
24  per  cent,  among  34  patients  not  receiving  serum  treatment;  of  8 
cases  due  to  Type  2,  all  recovered  but  two,  one  of  these  refusing  to  con- 
tinue the  treatment — a  mortality  of  25  per  cent,  as  compared  to  61  per 
cent,  among  13  patients  not  receiving  serum.  More  recent  reports 
indicate  that  the  percentage  of  predominating  types  of  pneumococci 
1  Jour.  Exper.  Med.,  1915,  22,  466. 


THE  SERUM  TREATMENT  OF  STREPTOCOCCUS  INFECTIONS    813 

producing  lobar  pneumonia  vary  in  different  years,  and  the  curative 
effect  of  the  sera  has  not  generally  been  found  as  promising  as  the  earlier 
results  indicated. 

Aside  from  this  decided  influence  upon  mortality,  the  general  effects 
of  the  serum  were  good.  In  10  cases  pneumococci  were  isolated  from 
the  blood  before  the  treatment  was  begun.  In  all  these  patients  the 
blood  had  become  sterile  after  the  first  treatment.  Following  the  in- 
jection of  serum  all  the  patients  seemed  to  feel  better,  and  in  a  number 
of  them  there  was  an  apparent  lessening  in  the  degree  of  intoxication. 
While  in  no  case  was  one  injection  sufficient  to  bring  about  a  crisis,  in 
all  except  the  fatal  cases  the  serum  had  apparently  an  ultimate  favorable 
effect,  lowering  the  temperature  and  shortening  the  course  of  the  disease. 


THE  SERUM  TREATMENT  OF  STREPTOCOCCUS  INFECTIONS 
The  acute  character  of  streptococcus  infections  and  their  relative 
frequency  and  severity  have  made  them  the  subject  of  numerous  efforts 
on  the  part  of  various  investigators  toward  developing  an  efficient  serum 
therapy.  To  Marmorek  belongs  the  credit  of  first  attempting,  in  1895, 
to  prepare  a  curative  serum  on  a  large  scale.  Since  then  Aronson, 
Tavel,  Krumbein,  Moser,  Meyer-Ruppel,  Menzer,  and  others  have  pre- 
pared immune  serums  with  various  cultures  and  according  to  various 
methods.  While  many  antistreptococcus  serums  will  show  undoubted 
protective  value,  especially  against  their  homologous  cultures  as  tested 
in  experimental  animals,  the  general  opinion  regarding  their  curative 
value  in  streptococcus  infections  of  man  have  been  conflictmg  and  as  a 
rule  unfavorable.  Occasionally  the  rapid  improvement  of  a  patient 
following  an  injection  of  the  serum  would  indicate  that  it  has  proved 
beneficial,  and  the  same  is  occasionally  true  of  a  particular  group  of 
infections  treated  with  a  specially  prepared  serum.  The  tendency  oi 
acute  streptococcus  infections  to  end  spontaneously  by  crisis  must, 
however,  be  borne  in  mind,  and  the  good  result  observed  in  individual 
cases  may  be  coincident  with,  rather  than  the  result  of,  the  administra- 
tion of  the  serum.  ^XT 

Several  causes  for  the  failure  of  antistreptococcus  serum  therapy 
are  now  understood,  and  if  these  can  be  eliminated,  the  value  of  this 
form  of  therapy  will  be  greatly  augmented. 

1.  The  serum  should  be  given  in  large  doses,  and  by  intramuscular  and 
intravenous  injection.  In  a  true  streptococcic  infection  the  cocci  are 
likely  at  some  time  to  be  found  in  the  blood-stream,  and  an  attempt  to 
destroy  these  organisms  or  to  limit  a  local  infection  by  injecting  10  c.c. 


814  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

of  serum  in  the  subcutaneous  tissues  is  almost  sure  to  result  in  failure. 
As  in  the  serum  treatment  of  pneumonia,  at  least  100  c.c.  of  serum 
should  be  given  intravenously  and  the  dose  repeated  if  necessary. 

2.  The  serum  should  be  used  as  early  in  the  disease  as  possible, 
instead  of  waiting  until  the  patient  has  become  moribund.     In  puerperal 
sepsis  and  scarlet  fever,  for  instance,  the  question  of  serum  therapy 
should  be  considered  early,  for  when  properly  administered,  the  serum 
will  at  least  do  no  harm  and  may  prove  efficacious.    In  order  to  determine 
the  value  of  the  serum  a  bacteriologic  diagnosis  should  always  be  at- 
tempted, especially  by  means  of  blood  cultures  obtained  by  placing  from 
2  to  5  c.c.  of  blood  in  a  flask  containing  at  least  100  c.c.  of  dextrose  broth 
just  prior  to  injecting  the  serum. 

3.  The  serum  should  be  polyvalent.     Marmorek  maintains  that  all 
streptococci  are  alike,  and  he  has,  accordingly,  prepared  his  serum  from 
a  single  highly  virulent  strain.     Other  investigators  question  this  asser- 
tion, and  at  present  the  consensus  of  opinion  is  agreed  that  streptococci 
from  different  infections,  such  as  puerperal  sepsis,  scarlet  fever,  ulcer- 
ative  endocarditis,  and  erysipelas,  exhibit  certain  immunologic  differ- 
ences, even  though  their  biologic  and  morphologic  characters  are  quite 
similar.     Thus  far  no  adequate  methods  for  differentiating  between 
these  organisms  have  been  discovered,  but  it  is  likely  that  future  re- 
searches will  show  that  streptococci  from  different  infections,  and  even 
from  cases  of  scarlet  fever,  possess  different  immunologic  characters 
similar  to  the  variations  observed  among  the  pneumococci  causing  lobar 
pneumonia.     If  this  is  found  to  be  true,  under  these  conditions,  a  sim- 
ilar serum  treatment,  while  complicated,  is  likely  to  prove  valuable  in 
the  treatment  of  streptococcus  infection.     For  the  treatment  of  strep- 
tococcus infection  in  scarlet  fever  the  serum  should  be  prepared  of  nu- 
merous strains  isolated  from  patients  having  this  disease.    What  has  just 
been  said  is  also  true  of  the  other  three  infections  so  frequently  strepto- 
coccal,  namely,  puerperal  sepsis,  phlegmonous  cellulitis,  and  ulcerative 
endocarditis.     If  not  these  four,  at  least  two  antistreptococcus  serums 
should  be  available :  one  for  scarlet  fever  and  the  other  for  other  infec- 
tions;  both,  and  especially  the  latter,  should  be  prepared  by  immuniz- 
ing horses  with  a  large  number  of  various  strains. 

Mode  of  Action  of  Antistreptococcus  Serum. — Virulent  strepto- 
cocci exert  a  powerful  negative  chemotactic  influence  upon  leukocytes, 
repelling  them  and  effectively  resisting  phagocytosis  for  varying  periods 
of  time.  The  early  researches  of  Bordet  showed  that  antistreptococcus 
serum  neutralizes  this  influence  and  promotes  phagocytosis.  Since 


THE  SERUM  TREATMENT  OF %  STREPTOCOCCUS  INFECTIONS    815 

then  numerous  investigators  have  supported  Bordet's  findings,  so  that 
it  may  be  accepted  as  true  that  one  of  the  chief  antibodies  in  antistrep- 
tococcic  serum  is  of  the  nature  of  a  bacteriotropin  or  immune  opsonin. 
A  potent  serum  also  contains  an  antitoxin,  as  may  be  shown  experi- 
mentally by  neutralization  of  the  hemotoxic  poison  of  streptococci, 
and  also  clinically,  when  the  rapid  subsidence  of  fever  and  general  im- 
provement of  the  patient  are  probably  due,  in  part,  to  neutralization  of 
streptococcal  toxins.  Thus  far  the  presence  of  bacteriolysins  has  not 
been  definitely  proved,  although  they  may  be  present  and  operative 
in  vivo.  I  have  found  that  antistreptococcus  serum  contains  an  anti- 
body capable  of  fixing  complement  with  streptococcus  antigens.1  It 
may  be  stated,  therefore,  that  the  action  of  antistreptococcus  serum  is 
dependent  primarily  upon  bacteriotropins,  and  secondarily  upon  anti- 
toxins and,  possibly,  bacteriolysins. 

Preparation  of  Antistreptococcus  Serum. — Some  differences  of  opinion  have 
been  expressed  regarding  the  advisability  of  passing  cultures  that  are  being  used 
for  purposes  of  immunization  through  a  lower  animal  in  order  to  increase  their  viru- 
lence. For  example,  the  unsatisfactory  results  that  have  followed  the  use  of  Mar- 
morek's  serum  have  been  ascribed  not  only  to  the  fact  that  it  is  monovalent,  but  also 
to  possible  alteration  of  the  strain  in  its  biologic  characteristics  by  animal  passage,  so 
that  its  virulence  for  the  human  being  was  diminished  or  lost,  and,  accordingly, 
while  the  antiserum  is  protective  for  the  animals  through  which  the  passage  has  been 
conducted,  it  is  inactive  for  the  human  being.  Tavel,  Krumbein,  and  Paltauf  have 
prepared  polyvalent  serums  with  different  strains  from  human  infections  without 
animal  passage.  Menzer  has  prepared  a  serum  with  strains  of  cocci  derived  from  acute 
rheumatic  fever,  and  Moser  with  strains  obtained  from  scarlet  fever,  which  have  also 
not  been  passed  through  animals.  Aronson  has  attempted  a  combined  procedure, 
making  use  of  passed  and  unpassed  cultures  conjointly,  and  this  appears  to  be  the 
method  of  choice.  In  other  words,  those  who  prepare  antistreptococcus  serums  should 
use  as  many  fresh  strains  as  possible,  and  hi  several  of  the  older  cultures  the  virulence 
should  be  increased  from  time  to  time  by  passage  through  animals. 

In  preparing  the  serum  young  and  healthy  horses  should  be  used.  The  injections 
should  first  commence  of  dead  cultures  given  subcutaneously,  then  of  autolysates, 
and  finally  of  living  cultures  administered  intravenously.  Occasionally  severe  local 
and  general  reactions  are  observed,  and  the  whole  procedure  should  be  conducted 
under  careful  supervision. 

First  Method. — Cultures  are  grown  on  a  solid  medium,  and  an  emulsion  and 
autolysate  prepared  as  described  for  immunizing  with  meningococci.  Begin  by 
injecting  subcutaneously  5  c.c.  of  emulsion  heated  to  60°  C.  for  an  hour,  and  in- 
creasing the  dose  each  week  by  5  c.c.  until  100  c.c.  are  given  at  one  time.  If  the 
reactions  are  mild  (general  and  local),  the  doses  may  be  increased  more  rapidly.  Then 
begin  with  2  c.c.  of  living  culture  and  increase  the  dose  each  week.  When  a  dose  of 
10  c.c.  is  reached,  inject  with  autolysate  and  living  cultures  alternately,  gradually 
increasing  the  dose  until  a  dose  of  50  c.c.  is  reached.  Living  cultures  are  then  given 
intravenously  and  the  dose  rapidly  increased  until  100  c.c.  and  more  are  given  at 

1  Arch,  of  Int.  Med.,  1912,  ix,  220. 


816  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

one  time.  Intravenous  injections  are  not  infrequently  tolerated  better  than  sub- 
cutaneous injections.  The  horses  may  be  bled  several  times"  during  the  course  of 
immunization  and  their  serums  tested.  When  the  serum  is  to  be  used  therapeutically, 
the  animals  should  not  be  bled  in  less  than  from  ten  to  fourteen  days  after  the  last 
injection  was  given.  In  view  of  the  large  doses  required,  a  concentrated  serum  is 
advisable  (Heinemann  and  Gatewood1)-  Since  trikresol  has  an  inhibiting  influence 
on  phagocytosis  (Weaver  and  Tunnicliff2),  the  minimal  quantity  (0.2  per  cent,  or 
less)  should  be  used,  or  preferably  no  preservative  at  all. 

Second  Method. — Kolle  prepares  a  polyvalent  serum  with  cultures  derived  from 
cases  of  erysipelas,  puerperal  sepsis,  scarlet  fever,  etc.  Their  virulence  is  increased 
from  time  to  time  by  passage  through  rabbits. 

Horses  are  used  for  immunization  purposes  and  all  injections  are  given  intra- 
venously at  intervals  of  a  week.  Cultures  are  grown  on  test-tubes  containing  a  solid 
medium,  and  immunization  is  started  with  half  a  culture,  heated.  The  dose  is 
increased  each  week  with  an  additional  culture  until  it  equals  16  cultures.  Then 
living  and  killed  cultures  are  mixed,  giving  in  one  week  2  living  and  14  killed  cultures 
and  so  on  until  10  living  and  6  killed  cultures  are  given  at  a  single  dose.  The  horses 
are  bled  two  weeks  after  the  last  dose  is  administered. 

Standardization  of  Antistreptococcus  Serum. — There  is  at.. present  no  single 
satisfactory  method  for  standardizing  these  serums,  although  a  satisfactory  method 
is  a  desideratum  for  testing  serums  placed  on  the  market.  In  order  to  obtain  an 
approximate  idea  as  to  the  value  of  a  serum,  the  following  tests  may  be  employed: 

1.  Protective  Value. — At  the  Serum  Institute  in  Vienna  a  passed  culture  (one 
used  in  the  process  of  immunization)  is  selected,  and  that  dose  which  will  kill  a  mouse 
at  the  expiration  of  or  just  preceding  the  end  of  four  days  is  regarded  as  a  single  lethal 
dose.    In  testing  an  antiserum  10  times  this  quantity  of  culture  is  used  with  decreasing 
doses  of  immune  serum  injected  twenty-four  hours  previously  or  simultaneously. 
A  normal  serum  is  one  of  which  0.01  c.c.  will  afford  protection,  and  1  c.c.  of  such  a 
serum  is  said  to  be  one  immunity  unit,  i.  e.,  it  affords  protection  against  1000  lethal 
doses  of  culture. 

2.  Bacteriotropic  Value. — The  technic  of  Wright  or  Neufeld  may  be  employed 
with  a  virulent  culture  and  human  leukocytes.    Weaver  and  Tunnicliff  have  observed 
better  results  when  using  one  part  of  immune  serum  reactivated  with  nine  parts  of 
fresh  guinea-pig  serum. 

3.  Complement-fixation  Tests. — These  tests  may  be  employed  with  the  bacterial 
emulsion  or  autolysate  used  in  immunization  as  the  antigen. 

The  serum  should  be  kept  in  a  cool,  dark  place.  After  a  few  months  it  loses 
some  of  its  protective  value,  and  much  of  it  on  the  market  is  worthless. 

Administration  of  Antistreptococcus  Serum. — It  must  be  emphasized 
here  that,  in  order  to  obtain  the  best  results,  antistreptococcus  serum  must 
be  given  intravenously.  In  an  adult  patient  with  a  severe  general  infec- 
tion from  30  to  100  c.c.  of  serum,  diluted  with  an  equal  amount  of  sterile 
normal  salt  solution,  may  be  given  in  one  dose,.  If  improvement  follows, 
subsequent  doses  should  be  given  subcutaneously  or  intramuscularly  in 
order  to  prolong  the  action  of  the  serum.  If  no  improvement  follows  in 
from  twelve  to  twenty-four  hours,  or  if  an  acute  exacerbation  sets  in,  a 
second  dose  should  be  given  intravenously.  Since  the  various  manu- 
1  Jour.  Infect.  Diseases,  1912,  x,  No.  3.  2  Jour.  Infect.  Diseases,  1911,  ix,  130. 


THE  SERUM  TREATMENT  OF  STREPTOCOCCUS  INFECTIONS    817 

facturers  use  different  cultures  in  the  preparation  of  these  serums,  it 
would  be  well  to  use  a  different  brand  of  serum  if  the  first  does  not  exert 
a  beneficial  effect.  In  patients  with  sever^  infections  the  activity  of  the 
serum  may  be  enhanced  by  adding,  just  tiefore  injection,  5  c.c.  of  fresh 
sterile  guinea-pig  serum  to  each  50  c.c.  of  the  immune  serum. 

In  the  treatment  of  localized  streptococcus  infections,  such  as  men- 
ingitis and  sinusitis,  it  may  be  well  to  mix  antistreptococcus  serum  with 
sodium  oleate  and  boric  acid,  as  previously  described. 

Value  of  Antistreptococcus  Serum. — Although  the  serum  has  been 
in  use  for  almost  twenty  years,  the  true  exact  value  of  the  remedy  has 
not  as  yet  been  estimated.  It  may  be  stated  that  a  carefully  prepared 
and  properly  administered  serum  will  do  no  harm  and  may  do  good,  and 
that  its  use  should  form  a  part  of  the  treatment  of  severe  streptococcal 
infections. 

In  some  cases  of  wound  infections  with  severe  cellulitis  and  septicemia 
the  serum  may  at  times  exert  a  most  pronounced  and  happy  effect.  In 
other  cases,  and  especially  in  those  in  whom  the  cocci  are  found  in  the 
blood,  repeated  injections  may  be  of  no  value. 

In  severe  anginose  or  malignant  scarlet  fever  large  doses  of  serum 
from  horses  especially  immunized  with  strains  of  streptococci  from 
scarlet-fever  patients  have,  on  the  whole,  yielded  favorable  results. 
Not  all  cases  of  severe  scarlet  fever,  however,  are  due  to  secondary 
streptococcal  infections :  those  patients  who  are  overwhelmed  and  pros- 
trated at  the  very  outset  are  probably  intoxicated  with  the  true  scar- 
latinal virus,  whatever  that  may  be,  and  such  cases  are  not  likely  to  be 
benefited  by  serum  treatment.  The  patients  most  likely  to  improve 
under  serum  therapy  are  those  who  become  severely  ill  after  the  onset 
of  the  disease  and  the  appearance  of  the  eruption. 

In  puerperal  sepsis  and  endocarditis  of  streptococcal  origin  the  results 
of  serum  treatment  have  not  been  uniform,  but  are  generally  unfavorable. 
If  serum  is  administered  at  all,  it  should  be  given  early,  in  large  doses, 
and  intravenously.  Not  all  cases  of  puerperal  sepsis  are  streptococcic, 
and  while  the  physician  may  not  be  justified  in  withholding  serum  until 
a  bacteriologic  diagnosis  has  been  made,  this  factor  must  be  considered 
when  estimating  the  value  of  a  serum. 

In  erysipelas  the  results  of  serum  treatment  have  been  very  indiffer- 
ent, but  may  at  times  prove  beneficial  in  severe  cases  when  administered 
in  large  doses,  and  the  same  may  be  said  of  bronchopneumonia,  laryngeal 
diphtheria,  small-pox,  and  tuberculosis. 
52 


818  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

THE  SERUM  TREATMENT  OF  GONOCOCCAL  INFECTIONS 
In  1906  Torrey  and  Rogers1  described  the  preparation  of  an  anti- 
gonococcus  serum  and  advocated  its  use  in  the  treatment  of  gonococcal 
infections,  and  especially  of  its  various  complications  and  metastases. 
In  the  following  year  these  observers  reported2  favorably  upon  the  re- 
sults of  serum  treatment  in  gonorrheal  arthritis,  and  to  a  lesser  extent  in 
infections  of  the  genito-urinary  organs.  Uhle  and  Mackinney3  used 
the  serum  in  the  treatment  of  23  cases  of  gonococcal  infection,  and  found 
it  beneficial  in  three  cases  of  gonorrheal  arthritis  and  in  one  of  myositis, 
whereas  in  epididymitis  and  urethritis  no  appreciable  effects  were  ob- 
served. Herbst  and  Belfield,4  Schmidt,5  and  Swinburne 6  agree  as  to  the 
value  of  the  serum  in  gonorrheal  arthritis,  whereas  in  other  complica- 
tions they  secured  somewhat  conflicting  results.  In  all  instances  the 
serum  was  used  in  relatively  small  doses,  namely,  2  c.c.,  injected  subcuta- 
neously  each  day  or  every  other  day,  the  number  of  injections  depend- 
ing on  the  clinical  condition  of  the  patient. 

Recently  Corbus 7  has  reported  more  favorable  results  in  the  treat-  • 
ment  of  24  cases  of  gonococcus  infection  by  using  larger  doses  of  serum — 
from  36  to  45  c.c. — injected  intramuscularly.  This  observer  advocates 
the  use  of  the  complement-fixation  test  as  a  reliable  guide  to  the  admin- 
istration of  the  serum :  the  more  intense  the  reaction,  the  more  efficient 
will  the  serum  prove;  if  the  reaction  is  negative,  the  serum  should  not 
be  used. 

Antigonococcus  serum  is  advocated  in  the  treatment  of  the  following 
conditions: 

1.  In  gonococcus  bacteremia. 

2.  In  those  infections  arising  by  extension  through  the  lymphatics 
or  the  circulatory  system,  as,  for  example,  arthritis,  iritis,  endocarditis, 
and  pleuritis. 

3.  In  those  acute  infections  arising  by  direct  extension  from  the 
urethra,  such  as  acute  prostatitis  and  epididymitis;   acute  orchitis  and 
probably  cystitis  in  the  male;  and  acute  salpingitis  in  the  female. 

The  serum  has  not  proved  of  value  in  the  treatment  of  gonorrheal 
conjunctivitis,  although  it  would  appear  advisable  to  employ  it  in  large 
doses  administered  intravenously  or  intramuscularly. 

1  Jour.  Amer.  Med.  Assoc.,  1906,  xlvi,  261,  273. 

2  Jour.  Amer.  Med.  Assoc.,  1907,  xlix,  918. 

3  Jour.  Amer.  Med.  Assoc.,  1908,  li,  105.    4  Illinois  Med.  Jour.,  1908,  xiii,  689. 

6  Therap.  Gaz.,  1909,  xxvi,  609.  •  Trans.  Amer.  Urol.  Assoc.,  1909,  iii,  170. 

7  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1462. 


THE    SERUM   TREATMENT    OF   STAPHYLOCOCCUS    INFECTIONS       819 

Preparation  pf  Antigonococcus  Serum. — Torrey's  serum  is  prepared  by 
immunizing  rams  with  gradually  increasing  intraperitoneal  doses  of  dead,  and  later 
of  living,  cultures  of  gonococci.  Larger  amounts  of  serum  may  be  secured  by  immu- 
nizing horses  according  to  the  methods  described  for  the  preparation  of  meningococcus 
and  streptococcus  immune  serums,  and  in  view  of  the  larger  doses  now  advocated, 
this  is  advisable.  This  serum  has  been  successfully  concentrated  in  the  same  manner 
as  is  diphtheria  antitoxin. 

Mode  of  Action  of  Antigonococcus  Serum. — According  to  Torrey, 
this  serum  is  largely  bacteriolytic  in  nature.  The  presence  of  antitoxins 
has  not  been  demonstrated;  small  amounts  of  bacteriotropins  are  pres- 
ent, so  that  a  potent  serum  probably  destroys  the  cocci  by  extracellular 
lysis  and  phagocytosis. 

Administration  of  Antigonococcus  Serum. — The  amount  of  serum 
used  and  the  method  of  inoculation  are  very  important  factors  in  the 
success  or  failure  of  the  treatment.  If  the  serum  is  used  at  all,  it  should 
be  given  in  large  doses.  The  original  method  of  giving  2  c.c.  subcu- 
taneously  has  been  found  inadequate  in  most  instances. 

In  acute  gonococcal  metastases  in  the  joints  or  in  the  endocardium 
or  other  serous  membrane,  from  30  to  50  c.c  of  serum  should  be  injected 
intravenously,  or  at  least  intramuscularly.  When  gonococci  are  found 
in  the  blood,  or  when  the  patient  is  profoundly  septic,  from  50  to  100  c.c. 
of  serum  should  be  given  intravenously.  In  epididymitis,  orchitis,  and 
other  local  complications  from  30.  to  50  c.c.  of  serum  should  be  given 
intramuscularly  or  intravenously.  Other  forms  of  treatment  should  be 
instituted  simultaneously.  If  necessary,  the  serum  injections  should  be 
repeated  in  twenty-four  hours,  and  if  a  good  primary  effect  follows  an 
intravenous  injection,  it  may  be  prolonged  by  one  or  more  subcutaneous 
or  intramuscular  injections  on  subsequent  days 


THE  SERUM  TREATMENT  OF  STAPHYLOCOCCUS  INFECTIONS 

While  several  attempts  have  been  made  to  treat  staphylococcus 
infections  with  an  immune  serum,  the  investigations  have  been  too  few 
and  too  brief  to  warrant  a  statement  in  regard  to  the  value  of  serum 
therapy  in  these  infections.  Thomas 1  prepared  a  serum  by  immunizing 
a  ram  with  18  different  strains  of  Staphylococcus  pyogenes  aureus,  and 
reported  good  results  in  the  treatment  of  28  cases  of  furunculosis  and 
carbuncles. 

It  is  probable  that,  with  more  extensive  use  of  antistaphylococcus 
serum,  its  value  will  be  proved,  especially  in  the  treatment  of  severe 
1  Jour.  Amer.  Med.  Assoc.,  1913,  Ix,  1070. 


820  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

furunculosis  of  infants  as  well  as  of  adults,  when  the  low  general  vitality 
of  the  patient  contraindicates  the  use  of  a  bacterial  vaccine.  With 
extended  use  it  may  also  be  found  to  be  of  benefit  in  staphylococcus 
bacteremia,  osteomyelitis,  arthritis,  carbuncle,  and  other  severe  in- 
fections. Following  recovery  or  relief  from  an  acute  infection  it  would 
seem  to  be  wise  actively  to  immunize  the  patient  with  a  vaccine,  or 
serum  and  vaccine  may  be  used  conjointly. 

The  activity  of  the  serum  is  probably  largely  dependent  upon  the 
presence  of  bacteriotropins  and  antitoxins,  the  former  promoting  phag- 
ocytosis and  the  latter  neutralizing  the  staphylolysins  or  hemotoxic 
poisons  produced  by  staphylococci. 

THE  SERUM  TREATMENT  OF  ANTHRAX 

Sclavo,1  Mendez,2  and  Deutsch3  have  prepared  anti-anthrax  serums 
by  immunizing  sheep,  goats,  asses,  and  horses  with  virulent  cultures  of 
anthrax  bacilli.  In  the  treatment  of  human  infections,  only  Sclavo's 
serum  has  been  used,  the  others  being  used  in  the  treatment  of  anthrax 
among  the  lower  animals.  An  anthrax  serum  prepared  by  American 
manufacturers  is  also  on  the  market. 

Sclavo's  serum  has  been  shown  to  possess  protective  and  therapeutic 
properties  in  experimental  infections,  and  favorable  results  have  been 
recorded  in  cases  of  human  anthrax.  According  to  Sclavo's  statistics, 
the  serum  has  reduced  the  average  mortality  of  anthrax  from  24  per 
cent,  to  5.3  per  cent.  Cigognani,  Legge,  Lockwood  and  Andrewes, 
Stretton  and  Mitchell,  and  others  have  reported  favorably  as  to  the 
value  of  the  serum. 

It  is  somewhat  difficult  to  prepare  a  potent  serum,  and  horses  should 
be  immunized  with  a  large  number  of  strains  from  human  infections  over 
a  long  period  of  time.  The  serum  is  probably  largely  bacteriotropic  and 
bacteriolytic  in  nature. 

Administration  of  Anti-anthrax  Serum. — In  the  Philadelphia  Hos- 
pital for  Contagious  Diseases  a  number  of  anthrax  cases  are  treated 
every  year.  Whenever  blood-cultures  have  revealed  the  presence  of  the 
bacilli  in  the  circulation,  the  patient  has  usually  succumbed  to  the  disease 
in  spite  of  serum  treatment;  the  doses  employed — 10  to  20  c.c. — may, 
however,  have  been  too  small.  I  would  recommend  the  following 
course  of  treatment  in  these  cases : 

1  Berl.  klin.  Wochenschr.,  1901,  18  and  19,  481,  520. 

2  Centralbl.  f .  Bakt.,  1899,  xxvi,  Nos.  21  and  22. 

3  Impfstoffe  u.  Sera,  Leipzig,  1903. 


THE  SERUM  TREATMENT  OF  TYPHOID  FEVER         821 

1.  If  the  lesion  is  relatively  small  and  accompanied  by  but  slight 
glandular  involvement,  with  little  or  no  evidences  of  toxemia,  a  blood 
culture  should  first  be  made  by  placing  from  2  to  5  c.c.  of  blood  in  a  flask 
of  neutral  bouillon,  followed  by  an  intravenous  or  intramuscular  injection 
of  from  20  to  50  c.c.  of  serum.     The  object  sought  is  to  introduce  serum 
before  the  lesion  is  handled,  and  the  blood  culture  is  the  best  indication 
for  subsequent  injections  of  serum  and  serves  as  a  guide  to  prognosis 

2.  The  lesion  should  then  be  excised,  leaving  a  wide  margin  so  as  to 
include  infected  lymphatic  channels.     It  should  be  handled  as  little  as 
possible.     The  wound  is  then  dusted  lightly  with  powdered  calomel 
and  next  heavily  powdered  with  ipecac.     Edema  soon  subsides,  and  the 
wound  usually  heals  rapidly,  with  surprisingly  little  scar-tissue  forma- 
tion.    If  the  edema  does  not  subside  and  the  infection  is  spreading  at  the 
margins  of  the  wound,  more  tissue  should  be  excised  or  multiple  local 
injections  with  phenol  and  anti-anthrax  serum  should  be  made. 

3.  The  blood  culture  may  be  examined  within  twenty-four  hours. 
If  anthrax  bacilli  are  present,  from  100  to  200  c.c.  of  serum  should  be 
given  intravenously,  the  injection  being  repeated  in  twenty-four  hours. 
Daily  blood  cultures  should  be  made,  and  the  serum  injections  con- 
tinued until  the  blood  becomes  sterile.     Salvarsan  may  also  be  injected 
intravenously.     In  our  experience,  cases  with  sterile  blood  cultures  have 
invariably  recovered. 

4.  In  internal  anthrax  all  the  physician  can  do  is  to  administer  large 
doses  of  the  serum  intravenously.     Salvarsan  may  also  be  tried.    (See 
Chapter  on  Chemotherapy.) 

. 
THE  SERUM  TREATMENT  OF  TYPHOID  FEVER 

The  serum  of  Chantemesse  is  the  only  serum  that  has  been  used  on  a 
large  scale  in  the  treatment  of  typhoid  fever  in  man. 

The  serum  is  derived  from  horses  that  have  been  immunized  for 
several  years  with  bouillon  filtrates  containing  typhoid  toxin,  chiefly 
endotoxin,  and  with  typhoid  bacilli.  Kraus  and  von  Stenitzer,  Meyer, 
Bergell,  and  Aronson  use  bouillon  filtrates  and  aqueous  bacterial  ex- 
tracts; Besredka  injects  dead  and  then  living  cultures;  MacFadyen 
uses  an  endotoxin  secured  by  breaking  up  cultures  frozen  at  very  low 
temperature.  It  would  appear  that  a  serum  should  be  bacteriolytic  and 
endotoxic,  and  this  probably  is  best  secured  by  prolonged  intravenous 
immunization  of  horses  with  a  large  number  of  dead  cultures  and  then 
with  autolysates  and  living  cultures  conjointly. 

According  to  Chantemesse,  the  subcutaneous  injection  of  a  few  drops 


822  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

of  his  serum  produces  leukocytosis  and  raises  the  opsonic  index  of  the 
patient's  serum.  He  emphasizes  the  fact  that  the  serum  should  be  given 
early, — before  the  seventh  day, — and  reports  that,  by  its  use,  the  mor- 
tality has  been  reduced  from  17  per  cent,  to  4.3  per  cent.  These  results 
have  not  been  generally  confirmed,  and  the  subject  is  still  sub  judice. 


THE  SERUM  TREATMENT  OF  PLAGUE 

Of  the  various  antipest  serums  that  have  been  prepared,  that  of 
Yersin  is  probably  best  known.  This  serum,  it  would  appear,  possesses 
some  prophylactic  and  therapeutic  value.  For  purposes  of  prophylaxis 
from  10  to  20  c.c.  of  serum  may  be  injected  subcutaneously  or  intra- 
venously; the  period  of  protection  is  short,  averaging  from  ten  to  four- 
teen days.  Combined  active  and  passive  immunization,  effected  by  means 
of  injections  of  a  pest  vaccine  and  an  antipest  serum,  will  probably  exert  a 
protective  action  of  several  months'  duration,  and  should  be  used  by  physi- 
cians, nurses,  and  others  during  epidemics  of  plague.  When  used  for 
therapeutic  purposes,  the  results  have  been  quite  variable.  If  serum  is 
used  in  the  treatment  of  plague,  it  should  be  given  as  early  as  possible, 
in  the  form  of  intravenous  or  intramuscular  injections  of  from  50  to  150 
c.c.,  if  this  amount  'is  available.  Injections  should  be  continued  at 
twelve-  to  twenty-four-hour  intervals  for  two  or  more  days  until  sup- 
puration has  been  controlled  and  the  disease  shows  signs  of  abating. 
The  Plague  Commission  of  India1  has  not  issued  very  favorable  reports 
upon  the  use  of  either  this  serum  or  that  of  Lustig. 

Chosksy,2  who  has  had  the  most  extensive  experience  with  the  serum 
treatment  of  plague,  emphasizes  the  necessity  of  treating  the  patient 
on  the  first  day.  He  advises  giving  100  c.c.  of  serum,  followed  by  a  second 
and  third  dose  at  intervals  of  six  to  eight  hours.  This  treatment  usually 
cut  short  the  attack  if  the  case  were  not  pneumonic,  malignant,  or  septi- 
cemic.  Of  400  cases  treated  with  serum  the  mortality  was  63.5  per  cent., 
while  in  200  controls  the  mortality  was  74  per  cent.  The  mortality  among 
cases  treated  with  serum  on  the  first  day  was  30  per  cent. ;  on  the  second 
day,  52  per  cent.,  with  a  gradual  increase  in  the  mortality  until  on  the 
seventh  day  the  mortality  was  100  per  cent.  Burnett3  has  reported  a 
mortality  of  29.7  per  cent,  among  cases  treated  with  serum  as  against 
a  mortality  of  73.9  per  cent,  treated  without  serum.  In  the  treatment 
of  pneumonic  plague  the  serum  has  given  no  favorable  results. 

r  Tj1  Britj,sh  Commission.    Seventh  Report  on  Plague  Investigation  in  India,  Jour. 
of  Hyg.,  Supplement  II,  1913,  326. 

2  Brit.  Med.  Jour.,  1908,  i,  1282. 

3  Report  on  Plague  in  Queensland,  1900-07,  Brisbane,  1907. 


THE  SERUM  TREATMENT  OF  CHOLERA  823 

(a)  In  addition  to  Yersin's  serum,  which  is  prepared  at  the  Pasteur  Institute  of 
Paris  by  immunizing  horses  with  dead  and  then  with  living  cultures  of  pest  bacilli, 
other  serums  have  been  prepared.  For  example: 

(6)  Kolle  immunizes  horses  with  intravenous  injections  of  heat-killed  cultures, 
beginning  with  Y±  agar  slant  culture  and  doubling  the  dose  each  week  until  15  cul- 
tures are  given  at  one  time.  The  horses  are  bled  fourteen  days  after  the  last  dose 
is  given. 

(c)  Lustig  immunizes  horses  with  pest-nucleoproteins,  obtained  by  breaking 
up  the  bacilli  with  1  per  cent,  of  potassium  hydroxid  and  precipitating  the  proteins 
with  acetic  acid.    These  are  then  suspended  in  sterile  normal  salt  solution,  as  in  the 
preparation  of  Lustig's  vaccine. 

(d)  Terni-Bandi  immunizes  donkeys  and  sheep  with  aggressins  obtained  by 
intraperitoneal  injection  of  guinea-pigs  with  pest  bacilli. 

(e)  Markl  immunizes  horses  with  nitrates  of  old  pest  bouillon  cultures.     He 
believes  that  the  value  of  pest  serum  is  largely  dependent  upon  antitoxins. 

The  serums  are  usually  tested  by  injecting  mice  with  lethal  doses  of  pest  culture 
and  decreasing  doses  of  antiserum.  The  agglutinin  content  may  also  be  measured. 
According  to  Strong  pest  immune  sera  do  not  contain  appreciable  amounts  of  bac- 
teriolysin,  but  are  largely  bacteriotropic  in  action.  Whenever  cultures  are  used  in 
immunization,  the  serum  should  always  be  cultured  carefully  and  tested  by  animal 
inoculation  to  guard  against  the  possibility  of  living  bacilli  being  present. 

i 

THE  SERUM  TREATMENT  OF  CHOLERA 

In  some  respects  cholera  would  seem  to  oe  due  mainly  to  a  toxin 
elaborated  by  the  bacilli  in  the  intestinal  tract  of  infected  persons, 
similar  to  the  action  of  the  toxin  of  the  Kruse-Shiga  type  of  dysentery 
bacillus.  Various  attempts  have  been  made  to  prepare  an  efficient 
anticholera  serum,  but  the  only  one  that  has  yielded  encouraging  results 
in  experimental  infections  as  well  as  in  cholera  of  human  beings  is  that 
prepared  by  Kraus.  This  serum  is  prepared  by  immunizing  horses 
with  a  true  toxin  derived  from  a  cholera-like  vibrio  isolated  by  Gottsch- 
lich  from  the  intestinal  contents  of  pilgrims  dying  at  El  Tor  from  a  dys- 
entery or  cholera-like  infection.  According  to  Kraus,  this  antiserum 
is  largely  antitoxic,  and  serves  to  neutralize  the  toxin  of  true  cholera 
more  effectively  than  does  the  antiserum  resulting  from  immunization 
with  cholera  cultures.  A  serum  that  is  antitoxic  and  is  obtained  by  pro- 
longed immunization  of  horses  by  intravenous  injections  with  dead  cul- 
tures of  cholera,  and  later  with  living  cultures,  bacterial  extracts,  and 
nitrates  of  old  bouillon  cultures  conjointly,  would  seem  to  be  a  desider- 
atum. 

Reports  from  Russia,  where  Kraus'  and  Kolle's  serums  have  been 

employed,  indicate  that  a  reduction  of  about  10  to  20  per  cent,  in  the 

mortality  has  been  accomplished.    Jegunoff's1  method  of  administering 

the  serum  seems  quite  rational,  and  consists  in  making  intravenous  in- 

1  Wien.  klin.  Wchnschr.,  1909,  No.  24. 


824  PASSIVE    IMMUNIZATION — SERUM   THERAPY 

jections  of  140  c.c.  of  serum  with  500  to  700  c.c.  of  sterile  salt  solution, 
followed  by  a  similar  or  slightly  lower  dosage  in  from  six  to  twenty-four 
hours  after  the  first  dose.  Twelve  patients  were  treated  in  this  way 
with  Kraus'  serum  with  a  mortality  of  25  per  cent.,  as  compared  with  a 
general  mortality  of  75  per  cent,  in  cases  receiving  no  serum.  The 
report  of  Hundogger1  upon  the  use  of  this  serum  is  unfavorable.  A 
thorough  review  of  the  literature  and  personal  investigations  of  Strong 
will  be  found  in  these  papers.2  The  intravenous  administration  of  salt 
solution  alone  has  proved  of  value  in  the  treatment  of  cholera,  and  it  is 
reasonable  to  assume  that  a  potent  serum  may  be  of  service  by  neutral- 
izing toxins,  destroying  the  bacilli,  and  at  least  furnishing  additional 
fluids  for  the  depleted  tissues  and  circulation. 

There  are  no  available  statistics  as  regards  the  prophylactic  value 
of  anticholera  serum,  but  combined  active  and  passive  immunization 
by  means  of  a  subcutaneous  injection  of  from  10  to  20  c.c.  of  serum, 
followed  by  three  doses  of  vaccine,  would  appear  to  be  a  rational  pro- 
cedure in  the  presence  of  an  epidemic  or  of  a  threatened  epidemic  of 
cholera. 

THE  SERUM  TREATMENT  OF  TUBERCULOSIS 

While  numerous  efforts  have  been  made  to  prepare  an  efficient  anti- 
tuberculosis  serum,  only  two — those  of  Maragliano  and  Marmorek — 
have  been  studied  and  are  familiar. 

Maragliano's  serum  is  prepared  by  immunizing  horses  for  from  four 
to  six  months  with  a  mixture  of  a  toxin  prepared  by  the  filtration  of  cul- 
tures only  a  few  days  old  and  concentrated  in  vacuo  at  a  temperature  of 
30°  C.,  mixed  with  that  obtained  by  aqueous  extraction  of  killed  virulent 
cultures  and  concentrated  by  heating  on  a  water-bath  at  100°  C.  for 
three  or  four  days.  Maragliano  assumes  that  the  antiserum  possesses 
antitoxic,  bactericidal,  and  agglutinating  properties.  One  cubic  centi- 
meter of  this  serum  is  injected  every  other  day  for  one  and  a  half  months. 
The  favorable  action  of  the  serum  is  reported  on,  especially  by  Mircoli 
and  other  Italian  physicians,  but  in  Germany  and  France  proof  of  its 
value  could  not  be  established. 

Marmorek's  serum  is  now  prepared  by  immunization  of  horses  with 
young  tubercle  bacilli,  whose  acid-fast  character  is  still  very  slight  or  en- 
tirely absent.  When  the  horses  have  attained  a  high  degree  of  immun- 
ity, they  receive  injections  of  various  strains  of  pure  cultures  of  strep- 

1  Wien.  klin.  Wchnschr.,  1909,  No.  52. 

2  Bull.  No.  16,  Bureau  of  Govt.  Lab.,  Manila,  1904;  ibid.,  No.  21:  Philadelphia  ' 
Jour.  Sci.,  1906,  i,  501;  ibid.,  1907,  ii,  413. 


THE    TREATMENT    OF    INFECTIOUS    DISEASES  825 

tococci  obtained  from  the  sputum  of  tuberculous  patients.  The  serum 
of  these  animals  is,  therefore,  antituberculous  and  also  antistreptococcic, 
and  is  serviceable  against  a  mixed  infection. 

The  serum  is  administered  daily,  either  by  subcutaneous  injection, 
in  doses  of  from  5  to  10  c.c.,  or  by  the  rectum  in  doses  of  from  10  to  20 
c.c.  The  latter  form  of  administration  is  quite  objectionable  to  most 
patients,  but  is  the  one  least  likely  to  produce  serum  sickness. 

While  this  serum  has  been  used  quite  extensively,  the  evidence  at 
present  is  too  conflicting  to  permit  definite  conclusions  to  be  drawn  as  to 
its  value  in  treatment.  It  would,  however,  seem  to  be  worthy  of  further 
trial  in  cases  of  localized  bone  and  joint  tuberculosis  and  in  the  incipient 
stage  of  pulmonary  tuberculosis.  Citron  recommends  its  use  in  patients 
who  evince  persistent  rise  of  temperature,  and  in  the  very  severe  but 
not  hopeless  cases  where  tuberculin  therapy  cannot  be  undertaken.  In 
some  of  these  cases  he  has  obtained  very  encouraging  results.  Citron 
occasionally  begins  with  the  serum  treatment,  and  later  combines  tuber- 
culin administration  with  it,  finally  omitting  the  serum  altogether. 


THE  TREATMENT   OF    INFECTIOUS    DISEASES    WITH    THE    SPECIFIC 
SERUM  OF  CONVALESCENTS  AND  WITH  NORMAL  SERUM 

On  the  basis  that  recovery  from  several  of  the  infectious  diseases 
confers  a  high  degree  of  immunity  due  presumably  to  the  presence  of 
specific  antibodies  in  the  body  fluids  and  particularly  the  blood,  numer- 
ous attempts  have  been  made  to  treat  certain  of  the  infectious  diseases, 
and  particularly  those  for  which  we  do  not  have  an  artificially  prepared 
serum,  with  serum  derived  from  convalescents. 

Scarlet  Fever. — Huber  and  Blumenthal,1  von  Leyden,2  Reiss  and 
Jungman,3  Koch,4  Rowe,5  Zingher,6  and  others  have  reported  favorably 
upon  the  treatment  of  scarlet  fever  and  particularly  severe  infections, 
with  injections  of  serum  from  persons  who  have  recovered  from  this 
disease.  Reiss  and  Jungman  obtained  the  serum  from  persons  about 
the  third  week  of  the  disease  and  injected  50  to  100  c.c.  intravenously. 
Koch  emphasizes  the  necessity  of  starting  the  treatment  as  early  in  the 
disease  as  possible  and  injecting  large  doses  of  the  serum  intravenously. 
Rowe  was  unable  to  convince  himself  that  there  were  any  different  effects 

1  Berl.  klin.  Wchnschr.,  1897,  xxxiv,  671. 

2  Deutsch.  Archiv.  f.  klin.  Med.,  1902,  Ixxiii,  616. 

3  Deutsch.  Archiv.  f.  klin.  Med.,  1912,  cxii,  70. 

4  Munch,  med.  Wchnschr.,  1913,  Ix,  2611;  Deutsch.  med.  Wchnschr.,  1915,  xli, 
372. 

5  Med.  Klinik.,  1913,  ix,  1978.  6  Jour.  Amer.  Med.  Assoc.,  1915,  65,  875. 


826  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

in  cases  treated  with  normal  and  with  convalescent  serum;  Koch  also 
suggests  that  the  convalescent  serum  be  reserved  for  the  gravest  cases, 
and  that  otherwise  normal  human  serum  be  used.  Zingher  has  used 
intramuscular  injections  of  whole  citrated  blood,  administering  from 
2  to  8  ounces  in  a  series  of  injections  at  close  intervals.  Normal  human 
blood  was  also  found  of  value  in  the  treatment  of  a  group  of  later  septic 
cases,  seen  from  the  fifth  to  the  eighth  day  of  the  disease.  It  is  apparent 
that  these  methods  of  treatment  are  worthy  of  further  trial,  and  particu- 
larly in  the  treatment  of  severe  or  anginose  cases  of  scarlet  fever. 

The  technic  is  very  simple  and  the  method  of  treatment  particularly 
applicable  in  hospitals  for  the  treatment  of  scarlet  fever.  Donors  are 
selected  after  the  second  week  of  the  disease,  and  preferably  from  among 
adolescents  or  adults  who  show  no  evidences  of  tuberculosis.  From  each 
person  30  to  100  c.c.  of  blood  may  be  obtained  under  aseptic  precautions 
and  the  serum  carefully  separated,  submitted  to  the  Wassermann  test, 
cultured  for  sterility,  and  preserved  with  0.2  per  cent,  tricresol  in  sterile 
containers  in  a  refrigerator.  In  young  children  an  intravenous  injection 
may  not  be  possible  unless  one  of  the  larger  veins,  as  the  external  jugular 
or  longitudinal  sinus,  is  selected.  It  is  advisable  to  inject  the  serum  as 
early  in  the  disease  as  possible  in  large  doses  (20  to  50  c.c.  for  a  child 
seven  years  of  age)  and,  preferably,  by  intravenous  injection.  The  in- 
jections appear  to  be  harmless  and  no  special  precautions  as  to  the 
presence  of  hemagglutinins  or  hemolysins  appear  necessary.  Otherwise 
the  serum  may  be  injected  intramuscularly  in  the  gluteal  region,  and  in 
the  absence  of  specific  convalescent  serum  normal  human  serum  collected 
in  the  same  manner  may  be  injected.  As  a  general  rule  three  or  four  in- 
jections are  required  at  intervals  of  six  to  twelve  hours  to  influence  the 
disease,  and  particularly  the  severe  infections. 

In  using  whole  blood  the  method  employed  by  Zingher  is  very  satis- 
factory. Two  c.c.  of  a  sterile  10  per  cent,  solution  of  sodium  citrate  in 
normal  salt  solution  is  placed  in  a  sterile  100-c.c.  bottle;  2  ounces  of 
blood  are  collected,  added,  and  briefly  shaken;  the  blood  is  now  ready 
for  intramuscular  injection.  Wassermann  reactions  should  be  made 
beforehand  on  all  possible  donors  so  that  the  proper  persons  may  be 
selected.  In  private  practice  the  physician  may  take  the  blood  from 
either  one  of  the  parents  or  close  relatives. 

Acute  Anterior  Poliomyelitis. — During  the  epidemic  of  acute  anterior 
poliomyelitis  in  1916  interest  was  renewed  in  the  treatment  of  the  disease 
with  intraspinal  and  intravenous  injections  of  serum  from  recovered 
and  normal  persons. 


THE    TREATMENT    OF    INFECTIOUS    DISEASES  827 

The  discovery,  almost  simultaneously  by  Flexner  and  Lewis1  and 
Landsteiner  and  Levaditi,2  of  the  filterable  nature  of  the  microorganism, 
or  virus  causing  the  disease,  was  quickly  followed  by  Flexner  and  Lewis/ 
observation  that  recovery  from  an  attack  of  experimental  poliomyelitis  af- 
forded protection  to  a  second  inoculation;  and  this,  in  turn,  was  followed 
by  the  detection  of  immunity  or  neutralizing  substances  in  the  blood 
serum  first  of  recovered  monkeys  and  then  of  recovered  human  beings 
by  Levaditi  and  Landsteiner,4  Romer  and  Joseph,5  Flexner  and  Lewis,6 
and  Anderson  and  Frost.7  Since  recovery  from  an  attack  of  poliomye- 
litis was  obviously  brought  about  through  a  process  of  immunization 
similar  to  that  in  other  infectious  diseases,  Flexner  and  Lewis8  endeav- 
ored to  prevent  the  development  of  the  infection  in  inoculated  monkeys 
through  the  administration  of  blood-serum  taken  (a)  from  recovered 
monkeys  and  (6)  from  recovered  human  beings.  The  results,  while  not 
constant  and  regular,  were  definite. 

These  experimental  results  were  at  once  utilized  as  a  basis  of  a  serum 
therapy  in  man  by  Netter  and  his  associates,9  who  have  reported  a  total 
of  34  cases  of  acute  poliomyelitis  which  they  have  treated  by  the  sub- 
dural  method  of  injecting  immune  serum.  They  have  undoubtedly 
established  the  fact  that,  as  in  the  monkey,  subdural  injections  intel- 
ligently carried  out  in  man  are  safe.  They  believe,  further,  that  they 
have  proved  them  to  be  definitely  beneficial  or  curative.  They  become 
increasingly  convinced  that  the  period  of  the  disease  at  which  the  injec- 
tions were  made  counted  vitally,  and  they  urged  that  the  injections 
should  be  made  as  early  as  possible  in  the  course  of  the  infection.  Fur- 
ther reports  by  Sophian,10  Alfaro  and  Hitce,11  Wells,12  Le  Boutillier,13 
Petty,14  and  Amoss  and  Chesney15  indicate  that  serum  taken  from  recently 
recovered  cases  of  poliomyelitis  may  be  employed  in  its  treatment  and 
probably  yields  the  best  results.  The  earlier  in  the  course  of  the  disease 
the  serum  is  employed  in  suitable  doses,  the  more  promise  there  is  of  bene- 
fit. The  action  of  the  serum  appears  to  be  more  precise  and  definite 
in  arresting  paralysis  than  in  rapidly  bringing  about  its  retrogression. 

1  Jour.  Amer.  Med.  Assoc.,  1909,  liii,  2095. 

2  Compt.  rend.  Soc.  biol.,  1909,  Ixvii,  592.       3  Jour.  Amer.  Med.  Assoc.,  1910,  liv,  45. 
4  Compt.  rend.  Soc.  biol.,  1910,  Ixviii,  311.       5  Munch,  med.  Woch.,  1910,  Ivii,  568. 

6  Jour.  Amer.  Med.  Assoc.,  1910,  liv,  1790. 

7  Jour.  Amer.  Med.  Assoc.,  1911,  Ivi,  663. 

8  Jour.  Amer.  Med.  Assoc.,  1910,  liv,  1780. 

9  Compt.  rend.  Soc.  biol.,  1911,  Ixx,  625;   Bull.  Acad.  med.,  1915,  Ixxiv,  series  3 
403;  Bull,  et  mem.  Soc.  med.  hop.,  Paris,  1916,  xl,  series  3,  299. 

10  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  426.  "  Semaine  Med.,  1915,  xxii,  211. 

12  Jour.  Amer.  Med.  Assoc.,  1916,  Ixvii,  1211.         13  Amer.  Jour.  Med.  Sci.,  1917. 
14  New  York  Med.  Jour.,  December  16, 1916.       l5  Jour.  Exper.  Med.,  1917,  xxv,  581. 


828  PASSIVE    IMMUNIZATION — SERUM    THERAPY 

Physicians  have  usually  administered  the  serum  by  intraspinal  injec- 
tion, but  the  experiments  of  Flexner  and  Amoss1  ascertained  that  an 
aseptic  meningitis  set  up  by  an  intraspinal  injection  of  any  serum  per- 
mits the  passage  of  immunity  principles  from  the  blood  into  the  cere- 
brospinal  fluid.  Therefore  the  intravenous  injection  of  serum  may 
influence  the  course  of  the  disease.  In  the  serum  treatment  of  epidemic 
poliomyelitis  Amoss  and  Chesney  state  that  the  following  conditions 
should  be  observed:  (1)  Early  and  prompt  diagnosis  and  treatment;  (2) 
intraspinal  injection  of  immune  serum;  (3)  intravenous  or  intramuscular 
injection  of  immune  serum;  (4)  the  serum  employed  should  be  collected 
from  cases  which  have  recently  passed  through  an  attack  of  poliomye- 
litis, as  it  is  to  be  supposed  that  the  serum  will  contain  a  greater  amount 
of  immune  principles  in  this  early  period  than  after  the  lapse  of  many 
years.  The  use  of  serum  from  recently  recovered  persons  otherwise 
healthy  involves  no  risk  in  transferring  the  microorganism  of  poliomye- 
litis, as  the  virus  has  never  been  detected  in  the  circulating  blood  of 
human  beings  even  in  the  first  days  of  the  disease. 

Blood  should  be  collected  under  aseptic  precautions  and  the  serum 
separated,  submitted  to  the  Wassermann  test,  cultured  for  sterility,  and 
kept  in  a  cold  place  preferably  without  the  addition  of  a  preservative. 
The  addition  of  0.2  per  cent,  tricresol  does  not  impair  the  curative  power. 
In  children  10  to  20  c.c.  of  serum  may  be  injected  intraspinally,  and 
20  to  30  c.c.  intramuscularly  or  intravenously.  Adults  should  receive 
larger  doses.  If  given  before  the  onset  of  paralysis  one  injection  may  be 
sufficient;  in  cases  in  which  some  degree  of  paralysis  develops  soon 
after  the  injection,  reinjection  twelve  to  twenty-four  hours  later  may  be 
advantageous. 

If  normal  human  serum  is  employed  it  should  be  injected  intra- 
spinally; the  good  effects  occasionally  observed  may  be  due  to  the  aseptic 
meningitis  produced  with  consequent  increased  permeability  of  the 
choroid  plexus,  and  the  passage  into  the  cerebrospinal  fluid  of  immunity 
principles  from  the  blood. 

Pneumonia.— Weissbecker,2  Huber  and  Blumenthal,3  and  others 
have  employed  convalescent  serum  in  the  treatment  of  lobar  pneu- 
monia with  apparently  encouraging  results.  If  this  form  of  therapy 
is  employed  it  is  necessary  to  ascertain  the  serologic  type  of  the  pneu- 
mococcus  producing  the  pneumonia  in  both  donor  and  recipient  and 
use  the  homologous  serum. 

1  Jour.  Exper.  Med.,  1914,  xx,  249;  ibid.,  1917,  xxv,  499. 

2  Ztsch.  f.  klin.  Med.,  1896,  xxx,  312;  ilrid.,  1897,  xxxii,  188. 

3  Berl.  klin.  Wchnschr.,  1897,  xxxiv,  671. 


CHAPTER  XXXI 
SERUM  THERAPY  (Continued) 

NORMAL  SERUM  THERAPY 

THE  field  of  serum  therapy  has  been  extended  in  recent  years  by  the 
successful  use  of  normal  serum  in  the  treatment  of  various  pathologic 
conditions,  particularly  the  hemorrhagic  diseases,  some  toxicoses  of  preg- 
nancy, and  certain  skin  affections. 

NORMAL  SERUM  m  THE  TREATMENT  OF  HEMORRHAGE 
Numerous  reports  by  Welch,1  John,2  Franz,3  Nohlia,4  Reichard,5 
Perkins,6  Claybrook,7  and  others  have  shown  that  injections  of  normal 
human,  horse,  or  rabbit  serum  are  of  considerable  value  in  the  treat- 
ment of  melena  neonatorum,  hemophilia,  purpura  hcemorrhagica,  hem- 
orrhagic retinitis,  intestinal  bleeding  in  typhoid  fever  and  in  connection 
with  cirrhosis  of  the  liver,  pulmonary  tuberculosis,  in  some  cases  of  uterine 
hemorrhage,  and  in  surgical  operations  upon  icteric  persons.  Barringer8 
has  reported  the  successful  treatment,  by.  injections  of  fresh  normal 
human  serum,  of  unilateral  kidney  hemorrhage  in  a  hemophiliac,  and 
advises  that  this  simple  treatment  should  be  tried  in  similar  cases  of 
varicose  veins  of  the  renal  papilla.  Levison9  has  reported  the  successful 
checking  of  hemorrhage  from  the  urinary  bladder  following  a  simple  op- 
eration by  performing  cystostomy,  removing  clots,  and  filling  the  bladder 
with  sterile  horse  serum. 

This  form  of  treatment  is  so  simple  and  has  been  so  successful  in  check- 
ing hemorrhage  in  melena  neonatorum  and  in  other  hemophilias  following 
injuries  or  operations  that  it  should  never  be  omitted. 

Defibrinated  human  blood,  human  serum,  or  the  serum  of  the  horse 

1  Amer.  Jour.  Obst.  and  Dis.  of  Women,  etc.,  1912,  Ixv,  No.  412. 

2  Munch,  med.  Wochenschr.,  1912,  lix,  No.  4. 

3  Munch,  med.  Wochenschr.,  1913,  lix,  2905. 

4  La  Presse  M6dicale,  1913,  xxi,  No.  20. 

6  Jour.  Amer.  Med.  Assoc.   1912,  lix,  1539. 


6  Jour.  Amer.  Med.  Assoc. 

7  Jour.  Amer.  Med.  Assoc. 

8  Jour.  Amer.  Med.  Assoc. 

9  Jour.  Amer.  Med.  Assoc. 


1912,  lix,  1539. 
1912,  lix,  1540. 

1912,  lix,  1538. 

1913,  Ix,  721. 
829 


830  SERUM    THERAPY 

and  rabbit  may  be  employed.    The  doses  advised  have  been  from  10  to 
20  c.c.  for  infants  and  children  and  from  20  to  50  c.c.  for  adults. 

The  technic  is  very  simple.  Sterile  normal  horse  serum  ready  for 
injection  may  be  purchased  in  the  open  market.  Human  serum  may  be 
secured  by  withdrawing  blood  into  large  centrifuge  tubes  (see  p.  33) 
and  allowing  the  serum  to  separate,  or  the  clot  may  be  broken  up  after 
an  hour  and  the  serum  secured  by  rapid  centrifugalization.  For  in- 
travenous medication  the  serum  should  be  free  from  particles  of  fibrin. 
Indeed,  the  whole  operation  may  be  conducted  at  the  bedside  by  with- 
drawing blood  from  the  donor  into  a  flask  containing  sterile  glass  beads, 
and  after  a  few  minutes  of  vigorous  shaking  the  defibrinated  blood  is 
injected  subcutaneously  or  intramuscularly.  Whenever  human  serum  or 
blood  is  used  and  time  permits,  a  Wassermann  reaction  should  be  per- 
formed beforehand,  and  it  should  be  determined,  by  hemolytic  and 
agglutination  tests,  that  the  donor's  serum  does  not  hemolyze  or  agglu- 
tinate the  recipient's  erythrocytes.  (See  p.  311.)  Of  course,  all  pro- 
cedures should  be  conducted  in  an  aseptic  manner. 

Blood  Transfusion. — Instead  of  injecting  serum  intravenously  or 
whole  blood  intramuscularly  in  the  treatment  of  the  anemia  following 
excessive  hemorrhage,  the  transfusion  of  whole  blood  is  frequently 
resorted  to  with  excellent  results.  Transfusion  has  been  employed  in 
(a)  the  treatment  of  severe  anemia  following  hemorrhage  from  injuries, 
operations,  and  child-birth;  (6)  in  the  severe  anemias  following  single 
or  repeated  hemorrhages  in  typhoid  fever,  gastric  ulcer,  carcinoma, 
tuberculosis,  or  similar  ulcerating  processes;  (c)  in  melena  neonatorum, 
hemophilia,  and  purpura  hemon-hagica;  (d)  in  the  treatment  of  perni- 
cious anemia  and  severe  anemias  of  the  chlorotic  type;  (e)  in  illumin- 
ating gas  poisoning,  and  (/)  in  the  passive  transfer  of  antibodies,  as  the 
transfusion  of  blood  from  a  typhoid  convalescent  to  a  person  suffering 
with  typhoid  fever.  The  best  results  have  been  observed  in  melena 
neonatorum,  in  which  disease  transfusion  often  acts  as  a  specific  remedy. 
In  hemophilia  the  bleeding  may  be  stopped,  but  the  disease  is  not  cured. 
In  gastric  ulcer  and  other  ulcerative  conditions  complicated  by  repeated 
hemorrhages  surgical  intervention  followed  immediately  by  transfusion 
has  yielded  successful  results.  In  pernicious  anemia  the  benefit  follow- 
ing transfusion  is  only  temporary;  repeated  transfusions  are  usually 
necessary,  and  splenectomy  followed  or  immediately  preceded  by  trans- 
fusion is  sometimes  employed. 

In  transfusion  the  person  yielding  the  blood  is  known  as  the  donor, 
while  the  person  receiving  it  is  known  as  the  recipient  or  donee.  In  select- 
ing a  donor  it  is  important  that  his  blood  be  compatible  with  that  of  the 


NORMAL    SERUM    THERAPY  831 

recipient.  Most  observers  are  now  agreed  that  preliminary  agglutination 
and  hemolysin  tests  should  be  conducted  in  order  to  avoid  the  occasional 
occurrence  of  severe  reactions  ascribed  to  intra vascular  agglutination, 
hemolysis,  or  both.  The  technic  of  these  tests  is  described  on  page  311. 
If  a  macroscopic  technic  is  employed  the  reactions  should  not  be  read 
as  negative  until  at  least  two  hours  have  elapsed.  If  there  is  sufficient 
time  a  Wassermann  reaction  should  be  conducted  with  the  serum  of 
the  donor. 

Methods. — A  large  number  of  methods  have  been  advocated  for 
the  transfusion  of  blood.  A  number  of  these  are  direct  methods  and  some- 
what intricate  and  painful  surgical  operations,  as  the  methods  of  Carrel,1 
Crile,2  Elsberg,3  Sauerbruch,4  Hartwell,5  Levin,6  Janeway,7  Soresi8 
and  McGrath,9  employing  sutures  and  cannulas  to  connect  the  vessel 
of  the  donor  with  the  vessel  of  the  recipient.  In  1909  Brewer  and 
Leggett10  used  simple  glass  tubes  coated  with  paraffin  with  satisfactory 
results;  Pope11  modified  this  method  by  using  a  rubber  tube  between 
two  glass  cannulas  inserted  in  the  vessels  of  donor  and  recipient;  Bern- 
heim12  used  two  silver  cannulas  in  the  same  manner,  one  cannula  fitting 
into  the  other  and  completing  the  connection.  In  all  of  these  methods, 
however,  accidents  were  likely  to  occur  which  interfered  with  the  success 
of  the  operation ;  furthermore,  it  was  difficult  to  maintain  an  aseptic  tech- 
nic and  the  amount  of  blood  transfused  could  not  be  exactly  determined. 

To  prevent  coagulation  and  permit  the  withdrawal  of  blood  and 
its  injection  into  the  recipient  without  direct  connection  between  the 
vessels  paraffin  coated  receptacles  were  employed  by  Curtis  and  David,13 
Kimpton  and  Brown,14  Satterlee  and  Hooper15  and  Percy.16 

As  early  as  1892  Ziemssen17  employed  a  syringe  method.  He  used 
a  number  of  needles  which  were  put  directly  into  the  veins  of  recipient 

1  Jour.  Exper.  Med.,  1907,  ix,  226;  ibid.,  1908,  x,  98;  ibid.,  1910,  xii,  460. 

2  Hemorrhage  and  Transfusion;  1908,  Appleton  &  Co. 

3  Jour.  Amer.  Med.  Assoc.,  1909,  lii,  887. 

4  Munch,  med.  Wchnschr.,  1915,  Ixii,  No.  45. 

5  Jour.  Amer.  Med.  Assoc.,  1909,  lii,  297. 

6  Ann.  of  Surg.,  1913,  May.  *  Ann.  of  Surg-)  1911>  lxiii>  720. 

8  Internal.  Cong,  of  Med.,  Budapest,  1909. 

9  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  40. 

10  Surg.  Gyn.,  and  Obstet.,  September,  1909. 

11  Jour.  Amer.  Med.  Assoc.,  1913,  Ix,  1284. 

12  Jour.  Amer.  Med.  Assoc.,  1912,  Iviii,  1007. 

13  Jour.  Amer.  Med.  Assoc.,  1911,  Ivi,  35. 

14  Jour.  Amer.  Med.  Assoc.,  1913,  Ixi,  117. 

15  Surg.,  Gyn.,  and  Obstet.,  1914,  xix,  235. 

16  Surg.,  Gyn.,  and  Obstet.,  1915,  xxi,  360. 

17  Munch,  med.  Wchnschr.,  1892,  No.  19,  323. 


832  SERUM   THERAPY 

and  donor;  three  syringes  of  25-c.c.  capacity  were  employed.  The 
sji-inge  was  filled  with  blood  from  the  donor,  which  was  then  injected 
directly  into  the  recipient.  While  this  injection  was  being  made  the 
second  syringe  was  being  filled.  When  the  first  syringe  was  emptied  of 
its  blood  it  was  immediately  washed  out  with  sterile  salt  solution  by  an 
assistant,  so  that  a  continuous  transfusion  was  conducted.  Lindeman1 
has  recently  elaborated  on  this  method,  and  similar  methods  have  been 
devised  by  Kush,2  Bernheim,3  Cooley  and  Vaughan,4  and  linger.5 

Still  later  herudin  and  sodium  citrate  were  employed  to  prevent  co- 
agulation and  permit  the  performance  of  transfusion  in  a  more  leisurely 
manner.  The  latter  substance  is  particularly  recommended  by  Hustin,6 
Dorrance,7  Wile,8  and  Lewisohn,9  and  is  being  widely  used  with  satis- 
factory results.  The  blood  of  the  donor  may  be  kept  in  a  refrigerator 
for  several  days  without  loosing  its  oxygen-carrying  capacity,  and  the 
injection  of  a  small  amount  of  sodium  citrate  (should  not  exceed  0.2  per 
cent.)  does  not  appear  to  lengthen  the  coagulation  time  of  the  blood 
of  the  recipient. 

As  previously  stated,  the  direct  methods,  consisting  in  suturing  artery 
to  vein  or  using  cannulas  and  tubes  to  connect  the  vessels  of  donor  and 
recipient,  are  being  discarded  in  favor  of  the  simpler  methods  which 
are  more  likely  to  be  successful  and  yield  excellent  results.  The  syringe 
methods  of  Bernheim10  and  linger11  are  particularly  satisfactory  for 
the  transfusion  of  whole  blood,  but  require  special  apparatus.  I  have 
used  successfully  the  original  method  of  Ziemssen  in  transfusing  adults 
when  proper  assistance  was  at  hand  for  rapid  work.  More  recently  I 
have  used  the  method  of  Lewisohn  and  Wile  with  success,  and  the  sim- 
plicity of  the  method  is  particularly  commendable.  According  to  Wile,  no 
unpleasant  symptoms  have  followed  the  injections  of  citrated  blood  in 
amounts  as  high  as  350  c.c.,  and  the  blood  may  be  kept  in  a  refrigerator 
for  three  to  five  days,  which  is  an  obvious  advantage  in  many  instances. 

Technic.—  The  technic  of  the  citrate  method  of  indirect  transfusion 
of  Wile  is  very  simple.  Blood  is  aspirated  from  a  vein  and  is  at  once 
well  mixed  with  sodium  citrate  in  10  per  cent,  solution  in  water  (steril- 

1  Jour.  Amer.  Med.  Assoc.,  1914,  Ixiii,  993;  ibid.,  1914,  Ixii,  1542. 

2  Jour.  Amer.  Med.  Assoc.,  1915,  Ixv,  1180. 

3  Jour.  Amer.  Med.  Assoc.,  1915,  Ixv,  1278. 

4  Jour.  Amer.  Med.  Assoc.,  1913,  Ix,  435. 

6  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  582;  ibid.,  1915,  Ixv,  1029. 

7  Ann.  et.  Bull.  Soc.  med.,  Bruxelles,  1914,  No.  4,  104. 

6  Penna.  Med.  Jour.,  1914,  xvii,  949.          »  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  425. 
»  Med.  Rec.,  January  23,  1915;  Surg.,  Gyn.,  and  Obstet.,  1915,  xxxi,  No.  1. 
"Loc.cit.  "Loc.cit. 


NORMAL    SERUM   THERAPY  833 

ized)  in  the  proportion  of  1  c.c.  of  solution  to  10  c.c.  of  blood.  Ex- 
ceptionally one  encounters  blood  which  requires  slightly  more  or  less 
citrate  proportionately.  Preliminary  tests,  0.1  c.c.  of  the  solution  to 
0.5,  1,  and  2  c.c.  of  blood,  respectively,  being  used,  enable  one  to  de- 
termine rapidly  the  requisite  proportions  in  any  individual  instance. 
If  the  mixture  is  made  in  the  syringe,  in  cases  in  which  not  more  than 
50  c.c.  are  to  be  transfused,  the  transfer  can  be  made  directly  from  donor 
to  recipient.  If  larger  amounts  are  to  be  used  the  blood  is  expelled 
into  a  flask  containing  the  sterile  solution  of  citrate,  from  which  the 
injecting  syringes  are  filled.  In  drawing  the  blood  it  is  convenient  to 
use  a  three-way  stop-cock  which  communicates  with  the  needle,  with 
a  10-c.c.  syringe  containing  the  citrate,  and  with  a  large  aspirating 
syringe. 

If  the  blood  is  to  be  injected  at  once,  I  heat  the  sodium  citrate  so- 
lution to  38°  C.;  if  the  blood-citrate  mixture  is  kept  in  the  refrigerator 
for  some  hours  before  injection  it  is  well  to  heat  the  mixture  before 
injecting  large  amounts  by  placing  the  flask  in  water  at  40°  C.  For 
injecting  large  amounts  (over  50  c.c.)  the  gravity  method  described 
on  page  743  and  illustrated  in  Fig.  140  may  be  used. 

In  giving  intravenous  injections  to  infants  the  longitudinal  sinus 
(Helmholz,1  Howard2)  or  external  jugular  vein  may  be  used.  To  reach 
the  sinus  the  needle  is  inserted  through  the  skin  in  the  superior  angle 
of  the  anterior  fontanel  at  an  angle  of  25  degrees  with  the  scalp.  A  20- 
or  30-c.c.  syringe  should  be  used. 

NORMAL  SERUM  IN  THE  TREATMENT  OF  THE  TOXICOSES  OF  PREGNANCY 
Feiux,  Freund,  Rongy,  and  others  have  found  injections  of  fresh 
normal  human  serum  from  pregnant  women,  serum  from  placental 
blood,  and  even  horse  serum  useful  in  the  treatment  of  the  vomiting  of 
pregnancy.  Freund  has  likewise  observed  that  injections  of  Ringer's 
and  Locke's  solutions  are  sometimes  efficacious;  he  has  found  injections 
of  serum  of  some  value  in  eclampsia,  and  tentatively  advises  its  use  in 
this  condition.  The  same  observer  has  employed  injections  of  normal 
serum  for  the  relief  of  the  itching  of  pregnancy,  and  reports  success. 
Similar  observations  have  been  made  by  Veiel3  and  Wolf,4  especially 
after  injections  of  serum  secured  from  other  healthy  pregnant  or  recently 
delivered  women. 

To  obtain  placental  serum,  the  following  technic  may  be  employed: 

1  Amer.  Jour.  Dis.  Children,  September,  1915,  194. 

2  Jour.  Amer.  Med.  Assoc.,  1915,  Ixv,  1365. 

3  Munch,  med.  Wchnschr.,  lix,  No.  35.          4  Berl.  klin.  Wchnschr.,  1913,.  1,  No.  36. 

53 


834  SERUM   THERAPY 

After  the  delivery  of  the  child  the  cord  is  sponged  with  bichlorid  solu- 
tion, cut,  and  the  maternal  end  bled  into  a  sterile,  wide-mouthed  flask. 
This  is  placed  in  the  refrigerator  until  there  is  complete  separation  of 
serum.  In  pipeting  off  the  serum  due  care  must  be  exercised  not  to 
include  corpuscles.  If  the  serum  is  not  perfectly  clear,  it  should  be 
centrifuged  with  aseptic  precautions.  It  is  well  to  have  each  serum 
tested  by  the  Wassermann  reaction  and  1  c.c.  cultured  in  100  c.c.  of 
glucose  bouillon  to  test  its  sterility.  The  serum  is  then  preserved  in 
bottles  or  vials,  with  the  addition  of  two  drops  of  5  per  cent,  phenol  to 
each  10  c.c.  of  serum. 

NORMAL  SERUM  IN  THE  TREATMENT  OF  SKIN  DISEASES 
In  addition  to  the  good  results  obtained  in  the  treatment  of  general 
pruritus  of  pregnancy  with  normal  serum,  Linser^Hench^and  Prsetorius3 
have  had  favorable  results  from  injections  of  normal  human  serum  or 
horse  serum  in  the  treatment  of  urticarial  and  chronic  obstinate  itching 
affections,  especially  senile  pruritus,  and  also  in  malignant  pemphigus. 
Even  better  results  have  been  observed  in  the  treatment  of  pemphigus, 
psoriasis,  and  other  skin  diseases  by  injections  of  the  patient's  own  serum. 
This  subject  will  be  referred  to  further  on. 

Mention  may  also  be  made  of  the  results  observed  in  the  treatment 
of  acute  and  chronic  nephritis  by  injections  of  blood-serum  from  the 
renal  vein  of  the  goat,  dog,  or  sheep.  Teissier4  was  probably  the  first 
to  apply  this  form  of  therapy,  and  reports  favorable  results  in  the  treat- 
ment of  seven  cases.  Spillman,5  Bisso,6  and  Dominquez7  have  also 
published  favorable  reports.  The  treatment  is  based  upon  the  assump- 
tion that  the  blood  in  the  renal  vein  contains  some  of  the  internal  secre- 
tion of  the  kidney,  which  acts  favorably  upon  the  liver  and  emunctories 
in  general.  This  method  of  treatment  has  been  advocated  by  the  pre- 
viously mentioned  observers  in  the  acute  exacerbations  of  chronic 
nephritis,  in  acute  or  chronic  nephritis  with  threatening  uremia,  and  in 
arterial  hypertension  presumably  of  renal  origin.  Amounts  of  serum 
ranging  from  10  to  50  c.c.  have  been  injected  subcutaneously,  and  re- 
peated on  several  days  or  every  other  day  until  several  doses  have  been 
given. 

1  Dermat.  Wochenschr.,  March  30,  1912. 

2  Munch,  med.  Wochenschr.,  1913,. lix,  No.  48. 

3  Munch,  med.  Wochenschr.,  1913,  Ix,  No.  16. 

4  Bull,  de  1'Acad.  de  MeU,  1908,  Ixxii,  No.  31. 
6  La  Presse  M&iicale,  1909,  xvii,  No.  86. 

6  Semana  Med.,  Buenos  Aires,  1912,  xix,  No.  7. 

7  Rev.  de  Med.  y  Cir.,  Havana,  1913,  xvii,  No.  24. 


AUTOSERUM   THERAPY  835 

AUTOSERUM  THERAPY 

A  number  of  observers  have  reported  favorable  results  following  the 
administration  of  the  patient's  own  serum  in  the  treatment  of  various 
diseases.  In  most  instances  serum  is  secured  by  withdrawing  blood  into 
a  sterile  container,  and  defibrinating  it  with  glass  beads;  this  is  followed 
by  thorough  centrifugalization,  or  the  serum  is  secured  after  the  blood 
has  been  allowed  to  coagulate  spontaneously.  In  other  instances  the 
serum  has  been  obtained  from  blisters  purposely  produced  by  the  appli- 
cation of  cantharides;  in  still  others,  and  especially  in  tuberculosis  of 
serous  membranes,  good  results  have  been  observed  to  follow  subcu- 
taneous injections  of  small  amounts  of  the  patient's  pleural  or  peritoneal 
fluid. 

AUTOSERUM  IN  THE  TREATMENT  OF  SKIN  DISEASES 
Spiethoff,1  Strumpke,2  Gottheil  and  Latenstein,3  Fox,4  and  other 
observers  have  secured  favorable  results  from  this  form  of  serum  therapy 
in  the  treatment  of  obstinate  and  chronic  dermatoses,  due  to  general, 
rather  than  to  local  causes,  in  which  the  usual  therapeutic  measures 
are  ineffectual  or  only  partially  successful,  as,  for  example,  psoriasis, 
dermatitis  herpetiformis,  pemphigus,  lichen  ruber,  lichen  planus,  urticaria, 
squamous  eczema,  etc.  Blood  is  withdrawn  from  the  patient  in  amounts 
of  from  50  to  100  c.c.  While  still  fresh,  the  serum  is  separated  and  in- 
jected intravenously  in  doses  of  from  30  to  40  c.c.  These  treatments  are 
repeated  from  two  to  six  times  at  intervals  of  from  three  to  five  days. 

In  collaboration  with  Schamberg  I  have  treated  several  cases  of 
psoriasis  with  autoserum,  injecting  the  serum  intravenously  in  doses  of 
20  c.c.  each  week  until  four  to  ten  injections  had  been  given.  In  none  wexe 
there  any  evidences  of  improvement.  Chrysarobin  ointment  was  not 
used  as  advised  by  Fox.  Ravitch5  has  also  reported  unfavorably  upon 
the  influence  of  autoserum  alone  in  the  treatment  of  psoriasis. 

AUTOSERUM  IN  THE  TREATMENT  OF  ACUTE  INFECTIOUS  DISEASES 
Favorable  results  have  also  been  observed  in  the  treatment  of  lep- 
rosy with  injections  of  serum  secured  by  raising  a  blister  with  cantharides. 
Similar  reports  have  been  made  by  Jez6  in  the  treatment  of  erysipelas, 

1  Med.  Klin.,  1913,  ix,  No.  24. 

*  Deut.  med.  Wchnschr.,  1913,  xxxix,  No.  30. 

3  Jour.  Amer.  Med.  Assoc.,  1914,  Ixiii,  1190. 

4  Jour.  Amer.  Med.  Assoc.,  1914,  63,  2190. 

5  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  1228. 

6  Wien.  klin.  Wchnschr.,  August  31,  1901. 


836  SERUM   THERAPY 

and  by  Mordinos1  in  that  of  typhoid  fever,  influenza,  and  Malta  fever. 
Other  observers  have  also  reported  good  results  following  the  injection 
of  from  5  to  10  c.c.  of  the  patient's  serum  in  the  treatment  of  gonorrheal 
arthritis,  typhoid  fever,  pneumonia,  and  other  infections.  Robertson2 
states  that  he  has  never  observed  the  slighest  influence  of  autoserum 
injections  in  the  treatment  of  typhoid  fever  and  pneumonia.  Palmer 
and  Secor3  have  reported  favorable  results  in  the  autoserum  treatment 
of  a  series  of  cases  of  pellagra,  and  Goodman4  in  the  treatment  of  chorea. 

AUTOSERUM  (SALVARSANIZED)  IN  THE  TREATMENT  OF  SYPHILIS  OF  THE  BRAIN 

AND  SPINAL  CORD 

The  treatment  of  syphilis  of  the  central  nervous  system  has  always 
been  unsatisfactory.  With  the  discovery  of  salvarsan  and  neosalvarsan, 
the  hope  was  fostered  that  these  remedies  would  prove  of  therapeutic 
value  in  the  treatment  of  tabes  dorsalis,  paresis,  and  cerebrospinal 
syphilis.  Experience  has  shown,  however,  that  while  the  progress  of 
tabes  dorsalis  may  be  arrested  in  the  early  stages  by  vigorous  treatment, 
in  paresis  the  prognosis  is  much  less  hopeful.  As  these  diseases  are  now 
known  to  be  truly  syphilitic,  the  presence  of  Treponema  pallidum  having 
been  actually  demonstrated  in  the  cerebral  cortex,  spinal  cord,  and 
cerebrospinal  fluid  by  Noguchi  and  Moore,  Nichols,  Graves,  Marie, 
Levaditi,  and  others,  the  cause  for  failure  in  the  treatment  of  these  in- 
fections must  be  ascribed  largely  to  the  fact  that  the  choroid  plexus 
filters  out  salvarsan  and  mercury,  as  well  as  antibodies,  and  prevents 
these  remedies  from  reaching  the  cerebrospinal  fluid,  just  as  it  prevents 
the  entrance  of  serum,  albumin,  sugar,  urea,  ammonia,  etc.  Although 
there  can  be  no  doubt  as  to  the  spirocheticidal  properties  of  salvarsan, 
and  as  to  its  ability  to  kill  the  treponema  in  the  tissues,  this  much-desired 
action  does  not  seem  to  occur  mainly  because  the  drug  cannot  gain  ac- 
cess to  the  parasites. 

Shortly  after  the  discovery  of  salvarsan  and  neosalvarsan  hope  was 
entertained  that  these  remedies;  when  injected  intraspinally,  would 
bring  the  drug  into  direct  contact  with  the  injected  tissues.  Investi- 
gations by  Wechselmann,5  Marinesco,6  and  Ellis  and  Swift7  have  shown, 

1  La  Presse  MSdicale,  1911,  xix,  No.  96,  1009. 

2  Personal  communication. 

3  Jour.  Amer.  Med.  Assoc.,  1915,  64,  1566. 

4  Archiv.  of  Pediat.,  1916,  xxxiii,  No.  9. 

5"Ehrlich-Hata  606,"  Berlin,  1911,  11.  Deut.  med.  Wchnschr.,  1912,  xxxviii, 
1446. 

6  Diatet.  u.  physik.  Therap.,  1913,  xvii,  194. 

7  Jour.  Exper.  Med.,  1913,  xviii,  428. 


AUTOSERUM   THERAPY  837 

however,  that  even  minute  quantities  of  either  drug  may  be  irritating 
and  may  prove  dangerous. 

Plaut1  showed  early  that  the  serum  of  patients  who  have  received 
salvarsan  exerts  a  definite  antisyphilitic  effect,  whereas  normal  serum 
displays  no  such  activity.  Meirowsky  and  Hartman2  and  Gibbs  and 
Calthrop3  observed  good  results  in  the  treatment  of  syphilis  from  sub- 
cutaneous injections  of  serum  from  other  patients  who  had  received 
salvarsan.  Swift  and  Ellis4  then  showed  that  serum  taken  from  a  pa- 
tient within  six  hours  after  the  salvarsan  was  injected  inhibited  the 
growth  of  Treponema  pallidum,  but  that  if  taken  before  the  salvarsan  was 
administered,  or  within  from  six  to  twenty-four  hours  after  treatment, 
there  was  no  inhibition.  These  last-named  observers  also  reported  bene- 
ficial effects  following  the  intraspinal  injection  of  salvarsanized  serum, 
with  but  slight  irritative  phenomena.  Further  experimental  studies  on 
the  spirocheticidal  activity  of  salvarsanized  serum  were  made  by  Gonder,5 
Castelli,6  and  especially  by  Swift  and  Ellis,7  the  last-named  investiga- 
tors also  noting  that  heating  the  serum  at  56°  C.  for  half  an  hour  mark- 
edly increased  its  activity,  which  was  due  in  part  to  the  destruction  of 
some  inhibiting  substance. 

Shortly  after  this  Swift  and  Ellis  8  published  a  report  of  the  treat- 
ment of  a  number  of  cases  of  tabes  dorsalis  and  other  syphilitic  infec- 
tions of  the  central  nervous  system  with  intraspinal  injections  of  sal- 
varsanized serum.  In  practically  all  these  cases  clinical  improvement 
was  observed,  with  total  or  partial  disappearance  of  the  positive  sero- 
biologic  findings  in  the  cerebrospinal  fluids.  Subsequent  reports  by 
Hough,9  McCaskey,10  Riggs,11  Pillsbury,12  Eskucken,13  Boggs  and  Snow- 
den,14  Litterer,15  McClure,16  Krida,17  Ayer,18  Draper,19  Smith,20  Dexter  and 

I  Deut.  med.  Wchnschr.,  1910,  xxxvi,  2237.  2  Med.  Klin.,  1910,  vi,  1572. 
3  Brit.  Med.  Jour.,  1911,  1,  360.                4  New  York  Med.  Jour.,  1912,  xcvi,  53. 

5  Zeitschr.  f.  Immunitatsf.,  Orig.,  1912,  xv,  57. 

6  Zeitschr.  f.  Chemotherap.,  Orig.,  1912,  1,  122  and  167. 

7  Jour.  Exp.  Med.,  1913,  xviii,  435.  8  Arch.  Int.  Med.,  1913,  xii,  331. 
9  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  183. 

10  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  187. 

II  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1888. 

12  Jour.  Amer.  Med.  Assoc.,  1914,  Ixvii,  15. 

13  Munch,  med.  Wchnschr.,  1914,  li,  No.  14. 

14  Arch.  Int.  Med.,  1914,  xiii,  970. 

15  The  Lancet-Clinic,  1915,  xciii,  359. 

18  Boston  Med.  and  Surg.  Jour.,  1914,  clxxi,  520. 

17  Albany  Med.  Ann.,  1914,  xxxv,  243. 

18  Boston  Med.  and  Surg.  Jour.,  1914,  clxx,  452. 

19  Archiv.  Int.  Med.,  1915,  xv,  16. 

20  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  1563. 


838  SERUM   THERAPY  . 

Cummer,1  Walker  and  Haller2  on  the  treatment  of  a  number  of  cases  of 
tabes  dorsalis,  paresis,  and  other  syphilitic  infections  of  the  central  nervous 
system  showed  that  this  method  of  treatment  possesses  distinct  value. 
Cutting  and  Mack,3  Myerson,4  and  Mapother  and  Beaton5  have  reported 
unfavorable  or  indifferent  results.  Intraspinal  injections  were  advocated 
by  Swift  and  Ellis  in  the  treatment  of  these  diseases  as  an  adjuvant  to 
intravenous  injections  of  salvarsan,  as  a  part  of  an  intensive  medi- 
cation aiming  to  bring  salvarsan  into  intimate  contact  with  the  para- 
sites in  the  most  direct  and  safest  manner.  While  this  form  of  treat- 
ment has,  in  a  large  percentage  of  cases,  effected  a  marked  improve- 
ment in  the  subjective  symptoms  and  has  modified  the  underlying 
pathologic  tissue  changes,  as  evidenced  by  the  disappearance  or  im- 
provement of  objective  signs  and  serobiologic  findings  in  the  cere- 
brospinal  fluid,  it  must  still  be  considered  in  the  experimental  stage, 
for  sufficient  time  has  not  yet  elapsed  to  permit  an  estimate  of  its  ulti- 
mate effect  to  be  made.  It  is  especially  indicated  in  early  and  incip- 
ient cases  of  syphilitic  infections  of  the  nervous  system.  It  is  self-evident 
that  it  cannot  be  expected  to  cure  cases  in  which  marked  tissue  destruc- 
tion has  occurred,  but  if  it  serves  to  cure  early  and  incipient  cases  of 
tabes  and  paresis,  or  at  least  tends  to  arrest  their  progress  and  possibly 
the  further  progress  of  more  chronic  cases,  and  gives  symptomatic 
relief,  then  this  mode  of  therapy  is  a  valuable  one.  At  present  it  is  ap- 
parent that,  in  the  hands  of  careful  and  competent  persons,  and  with  the 
strict  observance  of  the  original  technic,  the  method  is  relatively  devoid 
of  danger  and  constitutes  a  new,  rational,  and  valuable  addition  to  the 
treatment  of  diseases  that  may  otherwise  prove  intractable  to  the  ordin- 
ary antisyphilitic  measures. 

Technic. — From  0.6  to  0.9  gm.  of  salvarsan  or  neosalvarsan  is  in- 
jected intravenously.  One  hour  later  40  c.c.  of  blood  are  withdrawn 
directly  into  centrifuge  tubes  and  allowed  to  coagulate,  after  which  it  may 
be  centrifugalized.  The  following  day  12  c.c.  of  serum  are  pipeted  off  and 
diluted  with  18  c.c.  of  sterile  normal  salt  solution.  This  40  per  cent, 
serum  is  then  heated  at  56°  C.  for  one-half  hour.  After  lumbar  puncture 
the  cerebrospinal  fluid  is  withdrawn  until  the  pressure  is  reduced  to 
30  mm.  cerebrospinal  fluid  pressure.  The  barrel  of  a  20  c.c.  Luer  syr- 
inge (which  has  a  capacity  of  about  30  c.c.)  is  attached  to  the  needle  by 
means  of  a  rubber  tube  about  40  cm.  long.  The  tubing  is  allowed  to  fill 

1  Archiv.  Int.  Med.,  1916,  xvii,  82. 

2  Archiv.  Int.  Med.,  1916,  xviii,  376. 

3  Jour.  Amer.  Med.  Assoc.,  1914,  Ixvii,  903. 

4  Boston  Med.  and  Surg.  Jour.,  May  7,  1914. 
6  The  Lancet,  London,  1914,  clxxxvi,  1103. 


AUTOSERUM    THERAPY  839 

with  cerebrospinal  fluid,  so  that  no  air  will  be  injected.  The  serum  is 
then  poured  into  the  syringe,  and  permitted  to  flow  slowly  by  means  of 
gravity  into  the  subarachnoid  space.  At  times  it  is  necessary  to  insert 
the  plunger  of  the  syringe  to  inject  the  last  5  c.c.  of  fluid.  It  is  im- 
portant that  the  larger  part  of  the  serum  should  be  injected  by  gravity, 
and  if  the  rubber  tubing  is  not  more  than  40  cm.  long,  the  pressure  cannot 
be  higher  than  400  mm.  Usually  the  serum  flows  in  easily  even  under 
a  lower  pressure.  By  the  gravity  method  the  danger  of  suddenly  in- 
creasing the  intraspinous  pressure  to  the  danger-point,  such  as  might 
occur  with  rapid  injection  with  a  syringe,  is  avoided  (Swift  and  Ellis). 

The  method  of  injection  by  gravity  is  described  on  p.  746.  In  the 
absence  of  a  suitable  manometer  for  estimating  cerebrospinal  fluid  pres- 
sure the  blood-pressure  may  be  taken  as  a  guide;  in  any  event  the  serum 
should  be  injected  slowly. 

McCaskey  consumes  about  six  or  seven  minutes  in  administering 
the  salvarsan  intravenously,  and  advises  withdrawing  the  blood  twenty 
minutes  thereafter,  instead  of  waiting  an  hour  in  order  to  secure  a 
larger  quantity  of  the  drug  in  the  serum.  He  injects  15  c.c.  of  serum  in 
50  per  cent,  dilution  intraspinally,  and  has  not  observed  any  increased 
irritative  effects. 

Swift  and  Ellis  inject  the  serum  in  strengths  of  50  to  60  per  cent,  or 
even  higher  in  patients  who  do  not  exhibit  reactions  following  the  injec- 
tion of  40  per  cent,  serum.  Boggs  and  Snowden  withdraw  from  75  to 
100  c.c.  of  blood  one  hour  after  the  salvarsan  injection,  secure  the  serum, 
heat  it  at  56°  C.  for  one-half  hour,  and  inject  the  undiluted  serum  in  a 
dose  equal  to  the  amount  of  fluid  withdrawn,  whether  this  is  only  a  few 
or  as  much  as  30  c.c. 

The  method  employed  by  Marinesco  and  Minea,1  whereby  the 
patient's  serum  is  salvarsanized  in  vitro,  is  not  to  be  regarded  as  the 
same  as  the  method  of  Swift  and  Ellis.  The  first-named  investigators 
sought  to  administer  larger  doses  of  salvarsan  by  this  method,  but  in  a 
preliminary  report,  covering  the  treatment  of  20  cases  receiving  injec- 
tions every  seven  to  eight  days,  the  results  are  said  to  have  been  disap- 
pointing. The  recent  unfortunate  outcome  of  this  form  of  therapy  in 
the  County  Hospital  of  Los  Angeles  is  ascribed  to  the  oxidation  and 
consequent  toxicity  of  the  neosalvarsan  as  a  result  of  allowing  the  sal- 
varsanized serum  to  stand  for  twenty  hours  before  injecting  it. 

After-treatment. — The  patient  should  be  kept  in  bed  for  twenty-four 
hours  and  the  foot  of  the  bed  should  be  elevated  for  part  of  this  time. 
1  Bull,  de  1'Acad.  de  Med.,  1914,  Ixxvii,  No.  7. 


840  SERUM   THERAPY 

Usually  the  temperature  reaction  is  mild.  There  is  frequently  some 
pain  in  the  legs,  which  appears  a  few  hours  after  the  injection  is  given. 
The  pain  is  more  often  noticed  in  tabetics  than  in  patients  with  cerebro- 
spinal  syphilis.  It  can  usually  be  controlled  by  means  of  phenacetin 
and  codein,  but  occasionally  morphin  is  required. 

In  a  few  instances  violent  maniacal  symptoms  have  developed,  due 
possibly  to  the  direct  irritant  action  of  the  drug  on  the  tissues,  aided  by 
the  sudden  liberation  of  endotoxins  from  myriads  of  killed  spirochetes. 

Serobiologic  Findings  in  the  Cerebrospinal  Fluid. — In  all  cases  before 
the  treatment  is  undertaken  a  Wassermann  reaction  should  be  performed 
with  the  blood  and  cerebrospinal  fluid  of  the  patient.  A  total  cell  count 
should  also  be  made  with  a  specimen  of  fluid,  the  percentage  of  lympho- 
cytes ascertained,  and  a  Noguchi  butyric-acid-globulin  test  performed. 
Normally,  the  cells  number  about  8  per  cubic  millimeter  of  fluid  (counted 
with  a  Fuchs-Rosenthal  counting  chamber) ;  the  presence  of  more  than 
15  cells  in  this  quantity  of  fluid  may  be  regarded  as  bordering  on  the 
pathologic.  In  tabes  and  paresis  the  cells  may  vary  in  number  from 
50  to  more  than  100,  and  are  mostly  small  lymphocytes.  A  normal  cere- 
brospinal fluid  remains  clear  or  shows  a  faint  opalescence  when  tested 
by  the  Noguchi  butyric-acid  test.  In  tabes,  paresis,  etc.,  varying  de- 
grees of  cloudiness  and  the  presence  of  precipitates  are  observed. 

Repeating  the  Dose. — Usually  a  number  of  treatments  are  required, 
and  these  may  be  given  at  from  one  to  three  weeks'  intervals,  depending 
upon  the  condition  of  the  patient.  The  results  are  estimated  from  the 
subjective  and  objective  symptoms  and  from  an  examination  of  the 
cerebrospinal  fluid.  A  decrease  in  the  degree  of  positiveness  of  the 
Wassermann  reaction  and  diminution  in  cells  and  in  globulins  are  fav- 
orable signs.  Theoretically,  treatment  should  be  continued  until  the 
Wassermann  reaction  becomes  negative,  the  cells  reach  normal  propor- 
tions, and  the  globulins  show  no  increase.  Practically,  it  may  be  im- 
possible to  secure  these  results;  in  many  instances  the  Wassermann  re- 
action is  the  first  to  disappear.  A  total  cell  count  of  the  fluid  is  not  to 
be  depended  upon  without  a  differential  count  with  stained  smears,  for 
the  injections  may  produce  a  form  of  aseptic  meningitis,  accompanied  by 
an  outpouring  of  polynuclear  leukocytes.  This  subject  has  previously 
been  referred  to  in  a  consideration  of  the  serum  treatment  of  epidemic 
meningitis. 

Mechanism  of  Therapeutic  Action.— The  method  of  Swift  and  Ellis 
has  been  criticized  on  the  basis  that  it  was  unnecessary  because  the 
intravenous  injection  of  salvarsan  was  all-sufficient  in  its  action.  The 


AUTOSERUM   THERAPY  841 

authors  were  led  to  use  the  method,  however,  on  the  basis  of  the  investi- 
gations of  Adler,1  Hall,2  Swift,3  Camp,4  and  others,  indicating  that  no 
arsenic  or  only  mere  traces  appear  in  the  cerebrospinal  fluid  after  the 
intravenous  injection  of  salvarsan.  The  method  has  also  been  criticized 
on  the  basis  of  the  small  amount  of  arsenic  injected  intraspinally; 
Draper,5  Swift,6  and  Adler7  have  shown,  however,  that  blood  drawn 
a  short  time  after  rthe  intravenous  injection  of  salvarsan  contains  a 
definite  although  minute  amount  of  salvarsan.  It  would  appear, 
therefore,  that  in  this  method  of  treatment  salvarsan  is  introduced 
directly  into  the  cerebrospinal  fluid.  Beneficial  effects  may  also 
be  due  to  the  withdrawal  of  the  cerebrospinal  fluid  with  consequent 
increased  exudation  into  the  subarachnoid  space  of  serous  products 
from  the  blood-vessels  of  the  meninges.  Furthermore,  as  shown  by 
Flexner  and  Amoss,8  the  intraspinal  injection  of  most  any  fluid,  as 
sterile  salt  solution  or  normal  serum,  results  in  an  aseptic  meningitis 
and  increasing  the  permeability  of  the  choroid  plexus  and  meninges, 
and  in  this  manner  the  salvarsan  injected  intravenously  may  be  brought 
into  the  cerebrospinal  fluid,  the  intraspinal  injection  serving  primarily 
to  excite  the  aseptic  meningitis,  although  the  experiments  of  Stillman 
and  Swift9  showed  that  this  probability  is  rather  remote. 

AUTOSERUM  IN  THE  TREATMENT  OF  TUBERCULOSIS  OF  SEROUS  MEMBRANES 

Numerous  investigators,  as,  for  example,  Gilbert,10  Marcon,11 
Schnutgen,12Fishberg,13Pfender,14  Robertson,15  and  others,  have  reported 
favorable  results  in  the  treatment  of  tuberculous  pleurisy  with  effusion, 
cases  that  arise  either  insidiously  or  abruptly  with  pain  in  one  already 
tuberculous,  or  in  one  in  whom  tuberculosis  is  suspected,  following 
withdrawal  of  a  portion  of  the  fluid  and  immediate  injection  of  from  2 
to  5  c.c.  into  the  subcutaneous  tissues.  In  these  cases  there  is  usually 
a  sharp  reaction,  consisting  of  a  rise  in  temperature,  occasionally  ac- 

1  Boston  Med.  and  Surg.  Jour.,  1914,  clxxi,  900. 

2  Jour.  Amer.  Med.  Assoc.,  1915,  Ixiv,  1384. 

3  Jour.  Amer.  Med.  Assoc.,  1915,  Ixv,  209. 

4  The  Lancet-Clinic,  1915,  cxiii,  116. 

5  Archiv.  Int.  Hed.,  1915,  xv,  16. 

6  Loc.  cit.  7  Loc.  cit. 

8  Jour.  Exper.  Med.,  1917,  xxv,  499  and  525. 

9  Jour.  Exper.  Med.,  1915,  xxii,  286.  ">  Gaz.  des  Hopit.,  1894,  560. 

11  La  Presse  Medicale,  1909,  No.  71,  627. 

12  Berl.  klin.  Wochenschr.,  1909,  No.  3,  97. 

13  Jour.  Amer.  Med.  Assoc.,  1913,  Ix,  962. 

14  Wash.  Med.  Ann.,  1914,  xiii,  83.  l5  Personal  communication. 


842  SERUM   THERAPY 

companied  by  chill,  lassitude,  and,  in  the  majority  of  cases,  diuresis  or, 
more  rarely,  diarrhea,  followed  by  gradual  absorption  of  the  fluid  within 
the  following  few  days  up  to  two  or  three  weeks.  Fishberg  mentions  the 
disappearance  of  pain,  dyspnea,  and  prostration  within  two  or  three 
days  in  favorable  cases. 

It  is  difficult  to  state  whether  the  improvement  is  due  to  autotherapy 
or  simply  to  the  puncture  and  removal  of  so  much  fluid.  Eisner1  has 
seen  a  leukocytosis  follow  injection  of  serum  in  experimental  tuberculous 
infections  of  rabbits  and  guinea-pigs,  and  believes  that  this  explains  the 
good  results  in  this  particular  ^orm  of  therapy.  Zimmermann2  has  ex- 
pressed a  similar  opinion.  Other  investigators  assert  their  belief  in  the 
presence  of  aggressins,  bacteriolytic  amboceptors,  complements,  and 
endolysins  from  disintegrated  leukocytes  as  explaining  the  results.  It 
is  more  likely  that  these  fluids  contain  the  bacilli  or  their  products,  and 
constitute  a  form  of  vaccine  or  auto-tuberculin,  stimulating  body-cells 
to  produce  antibodies  largely  in  the  nature  of  bacteriotropins  and  bac- 
teriolysins.  Levy,  Valenzi  and  Ponzin,3  Szurek,4  and  Arnsperger5  are 
inclined  to  believe  that  the  beneficial  results  are  obtained  independently 
of  the  injections,  and  while  the  procedure  is  quite  generally  regarded  as 
perfectly  safe,  Jousset6  has  recorded  a  case  of  cold  abscess  following  an 
injection.  This  mode  of  treatment  seems  to  have  failed  in  about  10  to 
15  per  cent,  of  cases. 

The  technic  is  very  simple,  and  the  injections  may  be  given  by  any 
physician  who  can  make  an  ordinary  exploratory  puncture.  In  all  cases 
where  puncture  shows  the  presence  of  a  serous  fluid,  the  needle  should 
not  be  withdrawn  completely,  but  when  its  point  has  reached  the  sub- 
cutaneous tissues,  from  2  to  5  c.c.  should  be  injected  then  and  there.  In 
some  patients  it  will  be  necessary  to  repeat  the  treatment  several  times 
every  two  or  three  days  before  any  effect  becomes  evident.  Caforio 7 
has  reported  good  results  following  the  tapping  of  a  bilateral  hydrocek 
and  injection  of  a  portion  of  the  fluid  into  the  subcutaneous  tissues.  It 
would  appear  that  this  procedure  may  be  successful  in  hydrocele  of 
tuberculous  origin,  but  good  results  are  not  to  be  expected  in  cases  due 
to  contusion,  puncture  wounds,  gonorrheal  epididymitis,  or  orchitis. 

In  view  of  the  fact  that  the  fluid  may  contain  a  sufficient  number  of 
living  tubercle  bacilli  to  produce  secondary  infection,  it  would  appear 

1  Zeitschr.  f .  klin.  M«d.,  1912,  Ixxvi,  34. 

2  St.  Peters,  med.  Wchnschr.,  1909,  No.  34,  461. 

3  Bull.  et.  Mem.  de  la  Soc.  d.  Hop.  d.  Paris,  1910,  xxvii,  265. 

4  Med.  Klinik,  1909,  No.  44,  1665.          6  Therap.  d.  Gegenwart,  1911,  lii,  495. 
6  Arch.  G6n.  de  M6d.,  1912,  xci,  141.  7  Riform.  Med.,  1912,  xxviii,  1009. 


AUTOSERUM   THERAPY  843 

advisable  to  withdraw  fluid  into  an  equal  volume  of  2  per  cent,  sodium 
citrate  in  normal  salt  solution,  heat  at  60°  C.  for  one-half  to  one  hour, 
and  preserve  in  a  sterile  container  with  the  addition  of  a  few  drops  of  5 
per  cent,  phenol  until  ready  for  subcutaneous  injection  in  doses  of  from 
2  to  5  c.c. 

It  would  appear,  therefore,  that  reintroduction  into  the  body  of 
fluids  obtained  from  the  serous  cavities  (pleural,  peritoneal)  of  tuber- 
culous patients  may  be  of  value  in  treatment,  and,  as  a  general  rule,  the 
earlier  in  the  course  of  the  disease  that  the  autoserum  therapy  is  prac- 
tised, the  better  will  the  results  be.  A  similar  treatment  may  be  car- 
ried out  in  the  subacute  or  chronic  forms  of  tuberculous  meningitis. 
Fluid  is  to  be  collected  in  sodium  citrate  solution,  heated,  and  preserved 
with  phenol,  as  just  described.  The  dose  may  vary  from  0.5  to  2  c.c., 
according  to  age  and  clinical  conditions. 

AUTOSERUM  IN  THE  TREATMENT  OF  NON-TUBERCULOUS  EFFUSIONS 
In  pleural  and  peritoneal  effusions  of  renal,  cardiac,  or  hepatic  origin, 
this  method  of  autotherapy  has  generally  failed. 

Following  the  apparent  success  of  Hodenpyl1  in  the  treatment  of  a 
case  of  cancer  with  injections  of  the  patient's  ascitic  fluid,  the  autopsy 
subsequently  showing  the  presence  of  metastatic  cancer  not  demon- 
strable during  life,  Risely2  treated  65  cases  of  cancer  with  ascitic  fluids 
obtained  from  cancer  patients  in  all  stages  of  the  disease,  and  also  with 
various  normal  and  abnormal  body  fluids  from  other  than  cancerous 
conditions.  None  of  these  various  transudates  was  found  to  exert  any 
effect  in  retarding  the  growth  of  cancer  in  mice,  and  while  in  a  small  per- 
centage of  cases  large  doses  of  ascitic  fluid  of  cancerous  origin  may  re- 
lieve pain  and  retard  the  growth  of  the  cancer  for  from  one  to  five 
months,  no  permanent  effects,  either  preventing  or  checking  the  dis- 
ease, were  apparent. 

Leavy  and  Hastings8  and  Carter4  have  drawn  attention  to  the  ap- 
parent improvement  in  marasmic  infants  following  the  daily  injection, 
for  a  number  of  doses,  of  an  ounce  of  sterile  ascitic  fluid  (the  result  of 
cardiac  or  renal  disease)  into  the  subcutaneous  tissues  of  the  gluteal 
region  or  abdominal  wall.  As  these  fluids  are  non-toxic,  reinjection  is  a 
justifiable  procedure  after  the  fluids  have  been  subjected  to  the  Wasser- 
mann  reaction  and  to  careful  cultural  and  animal  injection  tests  to  prove 
their  sterility. 

1  Med.  Rec.,  1910,  Ixxvii,  359.          2  Jour.  Amer.  Med.  Assoc.,  1911,  Ivi,  1383. 

3  Bost.  Med.  and  Surg.  Jour.,  1910,  clxiii,  293. 

4  Amer.  Jour.  Med.  Sci.,  1911,  cxlii,  241. 


CHAPTER  XXXII 
CHEMOTHERAPY 

FROM  the  earliest  times,  healers  of  the  sick  have  sought  specific 
remedies  in  the  form  of  drugs  or  methods  that  would  always  and  un- 
failingly effect  the  cure  of  a  certain  disease,  and  indeed  this  search  has 
been  extended  for  a  single  remedy  that  will  cure  all  diseases,  regardless 
of  their  origin  and  nature.  To  this  end,  throughout  the  ages  experimen- 
tation, conscious  or  otherwise,  has  gone  hand  in  hand  with  medicine. 
Countless  substances  gathered  from  the  vegetable,  animal,  and  mineral 
kingdoms  have  been  experimented  with,  but  for  the  most  part  have  been 
discarded.  Despite  this  persistent  search  for  specifics,  until  compara- 
tively recent  times  but  two  remedies  have  been  found  worthy  of  being 
regarded  as  specifics,  namely,  cinchona  bark  for  malaria,  a  remedy  dis- 
covered by  the  South  American  Indians,  and  mercury  for  syphilis. 

With  discoveries  in  bacteriology  and  the  establishment  of  the  etio- 
logic  .relationship  of  various  microorganisms  to  certain  diseases,  test- 
tube  experiments  soon  demonstrated  that  certain  substances  could 
quickly  and  easily  destroy  these  microparasites.  It  was  apparent,  how- 
ever, that  in  the  animal  body  conditions  were  different;  here  the  germi- 
cide, even  when  administered  in  doses  sufficiently  large  to  prove  dangerous 
to  life,  usually  failed  to  kill  the  microorganisms.  The  exceptions  were 
quinin  and  mercury,  which  we  now  know  are  specifically  germicidal  for 
the  plasmodium  and  spirochete  respectively,  a  fact  that  was  suspected 
long  before  the  parasites  themselves  were  discovered. 

In  the  latter  part  of  the  eighties  it  was  discovered  that  the  blood 
possessed  germicidal  powers,  and  rapid  advances  were  soon  made  in  our 
knowledge  of  the  defensive  mechanism  of  the  animal  body,  and  the 
means  afforded  for  preventing  infection,  and  even  of  successfully  over- 
coming it  if,  by  any  chance,  microorganisms  passed  the  normal  barriers 
and  gained  a  foothold  on  the  tissues  proper.  Here  indeed  was  a  more 
or  less  specific  therapy  that  was  not  suspected  until  1894,  when  diph- 
theria antitoxin  was  discovered.  It  was  then  speedily  realized  that 
body-cells  could  be  made  to  produce  a  specific  remedy  for  a  certain  dis- 
ease, and  it  was  naturally  assumed  that  this  was  possible  for  all  those 

844 


PRINCIPLES    OF   CHEMOTHERAPY  845 

diseases  in  which  the  specific  microorganism  was  known  and  could  be 
cultivated. 

In  the  foregoing  chapters  we  have  reviewed  our  knowledge  of  the 
nature  of  these  specific  remedies  produced  by  the  body-cells  and  called 
antibodies.  In  Chapter  XXX  we  have  also  reviewed  the  application  of 
these  remedies  in  the  treatment  of  various  infections,  and  have  pointed 
out  their  limitations  and  the  difficulties  encountered  in  their  production. 
Naturally,  in  the  early  days  it  was  deemed  necessary  and  advisable  to 
utilize  a  lower  animal  for  the  manufacture  of  these  antibodies.  With 
the  discovery  of  a  means  of  attenuating  or  modifying  a  disease-producing 
parasite  it  was  found  possible  to  inject  these  vaccines  into  our  own 
bodies,  and  thus  stimulate  our  own  cells  to  produce  the  specific  anti- 
body— a  method  that  had  been  introduced  empirically  years  before  by 
Jenner  in  vaccination  against  smallpox. 

As  was  previously  stated,  scientists  have  long  believed  that  it  was 
possible  to  find  or  produce  chemical  substances  that  would  not  unite 
with  the  blood  albumin  or  body-cells,  but  would  have  a  highly  selective 
and  germicidal  action  on  microparasites  and  prove  capable  of  killing 
these  in  the  living  body.  Curiously  enough  Ehrlich,  to  whom  we  are  al- 
ready indebted  for  much  of  our  knowledge  of  the  intricate  problems 
of  cell  life,  and  especially  the  mechanism  of  their  defense  and  offense 
against  parasitic  invaders,  has  again  taken  the  lead  and  set  about  testing 
hundreds  of  compounds  in  a  painstaking,  logical,  and  scientific  manner, 
in  the  hope  of  finding  one  that  would  prove  most  germicidal  for  various 
trypanosomes  and  spirochetes,  and  at  the  same  time  be  least  toxic  for 
the  body-cells.  These  researches  finally  culminated  in  the  discovery 
of  the  arsenical  compound  now  popularly  known  as  salvarsan.  This 
discovery  constitutes  a  great  triumph  for  medical  science,  not  only  be- 
cause of  the  intrinsic  value  of  the  drug  in  the  treatment  and  cure  of 
syphilis  and  frambesia,  but  because  it  demonstrates  the  truth  of  a  prin- 
ciple, and  has  opened  up  vast  possibilities  for  future  investigation. 


PRINCIPLES  OF  CHEMOTHERAPY 

Organotropism  and  Parasitotropism. — The  guiding  principle  in 
chemotherapy  is  to  imitate  nature's  method  of  overcoming  an  infection 
by  the  aid  of  substances  that  destroy  the  microorganisms,  while  the 
body-cells  of  the  host  are  left  unharmed.  To  this  end  the  chemical 
agent  employed  must  possess  a  much  stronger  affinity  for  the  micro- 
organisms than  for  the  body-cells — to  quote  Ehrlich,  it  must  be  more 


846  CHEMOTHERAPY 

parasitotropic  than  organotropic.  If  a  given  chemical  agent  shows  a 
greater  affinity  for  the  albumins  of  the  body-juices  and  for  the  cells  than 
it  does  for  the  protoplasm  of  the  microparasites,  that  is,  if  it  is  more 
organotropic  than  parasitotropic,  it  is  evident  that  it  is  not  suitable 
for  therapeutic  purposes,  especially  if,  at  the  same  time,  the  toxic  dose 
for  the  host  should  be  smaller  than  that  for  the  microorganism. 

The  object  of  chemotherapeutic  research  is,  therefore,  to  discover  chem- 
icals that  have  a  greater  parasitotropic  than  organotropic  activity,  and  the 
greater  the  difference  between  these,  the  more  valuable  do  the  substances  be- 
come. 

There  is  only  one  way  of  determining  these  values,  and  that  is  by 
animal  experimentation.  Rats,  mice,  guinea-pigs,  rabbits,  and  fowls 
have  been  most  generally  employed  for  this  purpose,  and  most  work  so 
far  has  been  done  with  protozoan  parasites,  such  as  the  organism  of 
chicken  spirilloses,  the  spirillum  of  recurrent  fever,  various  trypano- 
somes,  and  the  spirochete  of  syphilis.  These  were  selected  because 
they  are  readily  found  in  the  blood  or  in  lesions,  and  are  rapidly  patho- 
genic. 

As  a  result  of  the  study  of  several  hundreds  of  different  products  by 
Ehrlich  and  his  collaborators  and  by  many  other  noted  investigators,  it 
was  found  that  there  are,  after  all,  comparatively  few  that  exert  a  para- 
sitotropic effect  in  animals;  these  are,  however,  well  characterized  chem- 
ically, and  have  been  classified  into  three  main  groups : 

1.  The  group  of  arsenical  compounds— arsenious  acid  (arsenic  tri- 
oxid),  atoxyl,  arsacetin,  arsenophenylglycin,  dioxydiamido-arsenobenzol 
dichlorhydrate  (popularly  known  as  "606,"  or  salvarsan),  and  various 
antimony  compounds. 

2.  Certain  azo-dyes  of  the  benzidin  group,  for  example,  trypan  red, 
trypan  blue,  and  trypan  violet. 

3.  The  group  of  basic  triphenylmethane  dyes,  such  as  parafuchsin, 
methyl-violet,  pyronin,  and  others. 

Newly  discovered  drugs  are  administered  in  increasing  amounts 
to  experimental  animals  until  the  toxic  dose  (dosis  lethalis),  the  tolerated 
dose  (dosis  tolerata),  and  the  curative  or  sterilizing  dose  (dosis  curativa 
or  sterilisana)  have  been  determined. 

While  chance  may  succeed  in  giving  us  a  drug  fulfilling  the  require- 
ments, so  far  it  has  had  little  influence,  and  it  is  difficult  to  realize  the 
tremendous  amount  of  work  done  by  Ehrlich  and  the  members  of  his 
institute  before  salvarsan  was  discovered.  The  following  table  illus- 
trates the  relation  of  the  tolerated  to  the  curative  doses  of  a  few  chem- 


PRINCIPLES  OF  CHEMOTHERAPY 


847 


ical  substances,  including  salvarsan,  given  by  intramuscular  injection, 
in  spirillosis  of  fowls,  and  illustrates  the  particular  value  of  "606,"  as 
dependent  upon  the  fact  that  it  is  highly  parasitotropic  in  an  amount 
that  is  far  below  the  organotropic  or  toxic  dose : 


CHEMICAL 

MAXIMUM 
TOLERATED 
DOSE 

STERILIZING 
OR  CURATIVE 
DOSE 

PROPORTION 

Atoxyl 

0.06 

0.03 

1:2 

Arsacctin 

0.1 

0.03 

1:3.3 

Arsenophenylglycin                                .... 

0.4 

0.12 

1:3.3 

Amidophenylarsenoxid  
Dioxydiamido-arsenobenzol  (salvarsan)  .  . 

0.03 
0.2 

0.0015 
0.0035 

1:20 
1:58 

Chemoreceptors. — Of  considerable  interest  in  this  connection  is  the 
question  of  how  substances  can  be  modified  in  order  to  render  them  pro- 
gressively more  parasitotropic  and  less  organotropic  in  action.  When 
ordinary  germicides,  such  as  phenol,  mercury  bichlorid,  formalin,  etc., 
are  added  to  suspensions  of  bacteria,  we  believe  that  the  latter  are  killed 
mainly  as  the  result  of  poisoning  of  their  protoplasm,  and  that  the 
chemical  substance  enters  into  chemical  union  with  the  bacterial  al- 
bumins by  direct  toxic  action,  accompanied  by  such  physical  changes  as 
that  of  coagulation,  and  alters  bacterial  metabolism  and  brings  about 
death  of  the  cells.  Such  germicides  do  not  appear  to  exert  any  selective 
action  on  any  particular  albumin.  When  mercury  bichlorid  is  added 
to  a  mixture  of  bacteria  in  a  serum,  many  of  the  microorganisms  may 
escape  destruction  through  the  formation  of  protective  envelops  of  an 
albuminate  of  mercury  formed  with  the  serum  albumins;  a  similar 
action  may  be  noted  with  phenol. 

In  chemotherapeutic  research,  therefore,  it  is  the  aim  to  start  with  a 
substance  that  primarily  shows  a  more  marked  affinity  for  the  proto- 
plasm of  the  parasite  than  it  does  for  the  body-cells,  and  then,  by  sub- 
tracting or  adding  to  the  molecule  or  by  inducing  an  intramolecular  re- 
arrangement, an  effort  is  made  to  develop  its  parasitotropic  action. 
The  question  then  arises,  does  the  increased  parasitotropism  of  the 
chemical  substance  depend  upon  its  higher  direct  and  simple  action 
upon  the  protoplasm  of  the  microparasite,  or  is  this  effect  to  be  explained 
upon  its  increased  combining  power  or  affinity  for  the  molecules  of  the 
parasites  or  other  cells  because  the  latter  are  provided  with  special  groups 
for  effecting  the  union?  Ehrlich  has  endeavored  to  answer  this  question 
by  maintaining  that  both  body-cells  and  microparasites  possess  special 
receptors  or  side-arms  by  which  chemical  substances  may  be  bound; 


848  CHEMOTHERAPY 

these  he  terms  chemoreceptors.  While  Ehrlich  originally  assumed  that 
the  so-called  side-arms  of  the  protoplasmic  molecule  served  primarily  for 
the  process  of  nutrition,  he  now  believes  that  these  special  chemore- 
ceptors do  exist.  He  suggests  that  they  may  probably  possess  a  less 
complex  structure,  similar  to  the  receptors  of  the  first  order  for  simple 
toxins,  that  they  are  more  firmly  attached  to  the  cell,  and  that,  accord- 
ingly, they  are  less  readily  cast  off,  thus  explaining  why  crystalline 
chemical  substances  are,  as  a  general  rule,  incapable  of  eliciting  the  pro- 
duction of  corresponding  antibodies.  This  theory  is  based  upon  the 
discovery  that  certain  strains  of  a  microparasite  develop  a  state  of 
" resistance"  or  "fastness"  to  a  particular  substance,  and  that  this  ac- 
quired characteristic  may  be  transmitted  from  generation  to  generation. 
This  subject  will  be  further  discussed  elsewhere. 

According  to  Ehrlich's  postulate,  therefore,  toxic  agents  cannot  act 
on  microorganisms  unless  they  are  fixed  by  suitable  receptors  (corpora 
non  agunt  nisi  fixata) .  This  conception  is  similar  in  every  way  to  his 
conception  of  the  processes  of  infection  and  immunity  and  the  develop- 
ment of  antibodies;  that  is,  the  toxic  agent  must  first  be  " fixed"  or 
"anchored"  to  the  molecule  of  a  cell  by  suitable  receptors  by  a  process 
of  chemical  interaction  before  damage  can  be  inflicted.  Chemoreceptors 
differ  from  other  receptors  in  being  more  firmly  attached  to  cells,  so 
that  while  the  cell  may  become  immune  to  the  toxic  effects  of  the  agent, 
the  blood-serum  may  not  contain  the  immune  bodies. 

When  arsenic  is  introduced  into  the  body,  it  is  "fixed"  by  the  re- 
ceptors of  certain  cells;  mercury  in  turn  is  fixed  by  other  receptors,  and 
so  on  through  the  list.  The  basic  principle  of  chemotherapy  is  that  it  is 
possible  to  produce  chemical  substances  that  carry  side-arms  capable  of  being 
fixed  by  microparasites,  and  to  a  much  less  extent  by  the  body-cells. 

It  is  not  necessary  that  the  whole  molecule  of  a  toxic  substance  possess 
a  combining  affinity  for  certain  receptors:  if  one  or  more  atom  groups 
becomes  attached,  it  is  presumed  that  it  carries  with  it  the  remainder 
of  the  molecule.  Moreover,  that  atom  group  that  is  anchored  or  is  re- 
sponsible for  the  anchorage  of  the  entire  molecule  need  not  possess  any 
of  the  properties  of  the  entire  molecule  or  of  any  part  thereof.  For  ex- 
ample, when  the  molecule  of  salvarsan  is  anchored  to  the  spirochete  of 
syphilis  by  its  OH  or  its  NH2  side-chain,  or  by  both,  the  spirochete  must 
later  contend  with  two  molecules  of  arsenic,  which,  being  in  a  trivalent 
condition,  can  exercise  its  toxic  effects  to  a  marked  degree  upon  the  para- 
site. The  side-arms  as  they  exist  in  salvarsan  are  much  more  readily 
taken  up  by  the  spirochete  than  by  the  body-cells,  which  is  shown  by 


PRINCIPLES  OF  CHEMOTHERAPY  849 

the  marked  difference  between  the  lethal  and  tolerated  doses  and  the 
curative  dose. 

Drug  "Fastness." — In  the  foregoing  chapters  we  have  sought  to 
emphasize  the  importance  of  the  microorganism  in  the  processes  of  in- 
fection and  immunity  from  the  standpoint  of  the  possibilities  of  these 
cells  immunizing  themselves  against  the  deleterious  agencies  of  the  host, 
and  particularly  the  antibodies,  as  explained  in  the  hypothesis  of  Welch. 
(See  p.  104.)  It  soon  appeared  that  the  problem  of  chemotherapy  was 
greatly  complicated  by  these  activities  on  the  part  of  the  parasite,  so 
that  the  hypothesis  appears  to  be  further  supported  as  a  result  of  chemo- 
therapeutic  studies.  If  the  dose  of  a  chemical  is  just  small  enough  to 
allow  a  few  microorganisms  to  escape,  these  immediately  fortify  ("  im- 
munize") themselves  against  the  drug  and  become  invulnerable  to  its 
effects.  It  was  found  that  these  microparasites  were  then  able  to  multi- 
ply, even  in  the  presence  of  the  drug,  and,  further,  that  this  property  of 
"drug  resistance"  was  transmitted  from  one  generation  to  another.  If, 
for  example,  the  trypanosomes  in  a  mouse  have  become  resistant  to 
trypan  red,  a  quantity  of  these  trypanosomes  may  be  inoculated  into 
another  mouse,  and  from  this  one  to  another,  and  so  on,  for  many  gen- 
erations, and  it  would  finally  be  found  that  the  trypanosomes  still. re- 
tained their  immunity  to  the  action  of  trypan  red. 

This  acquired  resistance  or  "fastness"  is  in  a  large  measure  specific. 
A  strain  of  trypanosomes  resistant  to  the  benzidin  dyes  is  non-resistant 
to  arsenic  and  the  triphenylmethane  dyes,  whereas  one  resistant  to  ar- 
senic is  not  resistant  to  the  dyes,  etc.  As  Levaditi  and  Fraser  have 
shown,  the  antibody-resistant  trypanosomes  do  not  anchor  the  antibody, 
hence  it  is  probable  that  the  analogy  holds  for  the  drug-resistant  micro- 
parasites. 

As  was  previously  stated,  Ehrlich  has  explained  this  phenomenon 
on  the  basis  of  chemoreceptors.  He  has  ascertained  that  "fastness" 
for  a  certain  chemical  agent  does  not  depend  on  atrophy  of  the  corres- 
ponding receptors,  but  upon  a  modification  in  their  structure,  as  is  evi- 
denced by  the  fact  that,  by  changing  the  structure  of  the  chemical,  it 
may  still  find  suitable  receptors  and  lead  to  the  destruction  of  the  para- 
site. For  example,  mice  that  have  been  infected  with  arsenic-fast  try- 
panosomes may  be  cured  by  an  injection  of  arsenophenylglycin,  even 
at  a  time  when  death  is  imminent.  It  would  appear,  therefore,  that 
the  arsenic-fast  receptors  of  the  trypanosomes  were  but  slightly  altered, 
and  were  still  capable  of  uniting  with  an  allied  product. 

This  factor  greatly  complicates  chemotherapy.     If,  for  instance,  the 

54 


850  CHEMOTHERAPY 

destruction  of  trypanosomes  by  an  arsenical  preparation  has  not  been 
complete,  and  if  the  antibodies  produced  by  the  body-cells  do  not  suc- 
ceed in  destroying  the  remainder,  there  is  a  strong  probability  that  a 
new  strain  will  now  develop  that  will  be  resistant  not  only  to  the  par- 
ticular arsenical  preparation  used,  but  will  also  be  proof  against  the 
serum  antibodies.  This  new  strain  may  now  cause  a  relapse  of  the 
disease.  Another  chemical  is  now  injected,  but  if  this  likewise  fails  to 
kill  all  the  trypanosomes,  the  remainder  will  generate  still  another 
strain  "resistant"  to  the  preparation  and  the  antibodies.  This  may 
continue  as  long  as  the  parasite  is  able  to  produce  new  receptors;  when 
this  limit  is  reached,  its  nutrition  will  be  impaired  and  the  serum  anti- 
bodies, alone  or  aided  by  another  chemical  substance,  may  finally 
destroy  all  trypanosomes,  the  infection  "dying  out/'  so  to  speak,  or 
proving  completely  vulnerable  to  a  chemical  agent. 

These  considerations  have  an  actual  experimental  basis,  and  natural 
examples  of  acquired  serum-resistance  or  "fastness"  are  to  be  found  in 
relapsing  fever,  syphilis,  sleeping  sickness,  and  possibly  malaria.  No- 
guchi  and  Akatsu1  have  recently  succeeded  in  rendering  different  strains 
of  spirochetes  "drug-fast"  to  various  compounds  of  arsenic  and  mercury 
in  vitro.  In  relapsing  fever  the  clinical  course  of  the  disease  would  indi- 
dicate  that  only  three  or  four  serum-fast  strains  can  be  produced,  and  we 
accordingly  find  that,  after  a  patient  has  withstood  a  number  of  relapses 
corresponding  to  the  number  of  antibody-fast  strains  that  the  spirillum 
may  produce,  spontaneous  recovery  occurs,  there  being  then  antibodies 
that  the  strain  cannot  resist,  and  the  infection  "dies  out,"  due  in  part  to 
destruction  of  the  antibodies  and  in  part  to  starvation  of  the  parasite 
because  the  number  of  receptors  is  insufficient  to  carry  on  nutrition. 
In  the  mean  time,  however,  the  patient  may  succumb  to  the  disease 
before  the  spirillum  has  "played  its  last  card." 

In  syphilis  conditions  are  different,  and  the  phenomenon  of  "resis- 
tant races"  further  explains  many  conditions  not  previously  understood. 
The  spirochete  is  apparently  capable  of  repairing  its  receptors  to  a  re- 
markable degree,  especially  after  injury  received  from  the  antibodies  pro- 
duced by  body-cells,  and,  further,  is  able  to  maintain  its  nutrition  with 
a  number  of  different  food-stuffs,  so  that  in  the  untreated  person  re- 
lapse follows  relapse,  the  offensive  forces  of  the  spirochete  being  held  in 
abeyance  by  the  defenses  of  the  host  over  long  periods  of  time  (latent 
periods),  but  the  vital  parts  of  the  host  being  gradually  damaged  and 
the  defenses  weakened  so  that  death  or  serious  symptoms  may  supervene 
1  Jour.  Exper.  Med.,  1917,  xxv,  349  and  363. 


PRINCIPLES    OF    CHEMOTHERAPY 


851 


long  before  the  disease  has  "worn  itself  out,  "if,  indeed,  this  ever  occurs 
in  syphilis. 

The  theory  of  chemoreceptors  affords  an  explanation  of  this  phe- 
nomenon, just  as  the  side-chain  theory  affords  an  explanation  of  the  for- 
mation of  cellular  antibodies  and  the  processes  of  immunity.  In  adopt- 
ing it  we  must  be  prepared  to  believe  that  the  number  of  receptors  and 
the  possibilities  of  their  repair  or  modification  are  practically  unlimited, 
as  evidenced  by  the  large 
number  of  substances  capable 
of  exerting  toxic  action.  Syph- 
ilis, for  example,  is  probably 
a  disease  of  great  antiquity, 
handed  down  from  person  to 
person,  from  generation  to  gen- 
eration, during  which  trans- 
mission the  receptors  of  the 
spirochete  must  have  under- 
gone innumerable  transforma- 
tions and  alterations,  yet  find- 
ing suitable  receptors  in  the 
cells  of  practically  all  non- 
syphilitic  persons,  although  it 
is  now  apparent  that  strains 
have  gradually  been  evolved 
that  possess  receptors  with 

marked  affinity  for  certain  tissues,  as,  for  example,  those  for  the  central 
nervous  system,  cardiovascular  system,  etc. 

Therapia  Magna  Sterilisans. — As  the  development  of  resistant 
strains  is  thus  one  of  t?he  possibilities  and  impediments  to  successful 
specific  therapy,  whether  with  chemicals  (chemotherapy)  or  with  anti- 
bodies (serum  therapy),  our  efforts  should  be  directed  toward  discover- 
ing chemical  substances  and  a  method  of  administration  that  will  com- 
pletely sterilize  the  individual  at  one  time  (Ehrlich's  therapia  magna 
sterilisans).  This  possibility  has  been  amply  demonstrated  experi- 
mentally by  Ehrlich,  and  it  has  occasionally  been  accomplished  ia  the 
early  stages  of  human  syphilis  by  means  of  salvarsan  administered  early 
in  the  proper  manner  and  in  correct  dosage,  but,  unfortunately,  it  is 
not  true  of  the  majority  of  cases,  the  difficulties  increasing  with  the 
duration  of  the  infection.  Fortunately,  the  investigations  of  Margulies 
1  "Die  Behandlung  der  Syphilis"  (Konigsberg  Versammlung),  Leipzig,  1910, 930. 


FIG.  144. — BLOOD  OF  A  RAT  INFECTED  WITH 

SPIROCELETA  RECURRENTIS. 

(Drawing  made  from  dark-field  illumination 

just  before  administration  of  salvarsan.) 


852 


CHEMOTHERAPY 


have  shown  that  while  resistant  races  of  trypanosomes  are  easily  evolved, 
the  evidence  is  entirely  against  the  probability  of  the  development  of  arsenic- 
resistant  syphilitic  spirochetes  as  the  result  of  prolonged  treatment  'with  non- 
sterilizing  doses  of  salvarsan.  Akatsu  and  Noguchi1  have,  however,  in- 
creased the  tolerance  of  Treponema  pallidum  to  salvarsan  and  neosal- 
varsan  five  and-one-half  times  their  original  mark  by  cultivating  the 
spirochete  in  culture-media  containing  these  drugs. 

With  this  brief  discussion  of  the  primary  principles  of  chemotherapy, 
which  is  really  a  very  ancient  method  of  treatment  and  dates  from  the 

time  that  chemicals  were  first 
used  for  treating  the  sick, 
but  which  has  now  emerged 
from  the  darkness  of  pure  em- 
piricism into  the  light  of  a 
science,  we  shall  pass  on  to  a 
consideration  of  salvarsan  and 
neosalvarsan.  These  two  sub- 
stances were  not  discovered  as 
the  result  of  accident,  but 
were  the  outcome  of  exact 
knowledge,  logical  thinking, 
and  carefully  planned  experi- 
mentation. It  is  impossible 
to  tell  what  these  discoveries 
presage;  certainly  they  open 
up  vast  possibilities  in  the 

development  of  a  specific  therapy  for  all  infections.  One  fact  is  cer- 
tain: that  while  chance  must  ever  play  some  role,  future  discoveries 
will  probably  result  only  from  prolonged,  patient,  and  laborious  study. 


FIG.  145. — BLOOD  OF  SAME  RAT  EIGH- 
TEEN HOURS  AFTER  INTRAVENOUS  INJECTION 
OF  0.0008  MG.  SALVARSAN. 


SALVARSAN  AND  NEOSALVARSAN  IN  THE  TREATMENT  OF  SYPHILIS 

Historic. — The  administration  of  arsenic  in  protozoan  infections  has 
long  been  a  recognized  method  of  treatment.  The  organic  compound 
of  arsenic  known  as  atoxyl  (the  sodium  salt  of  para-aminophenylarsenic 
acid)  was  first  used  in  the  treatment  of  trypanosomiasis,  and  although 
this  drug  did  not  produce  the  results  that  were  anticipated,  it  formed  the 
starting-point  for  important  researches  in  the  preparation  of  organic 
compounds  of  arsenic  and  their  use  in  protozoan  infections.  On  the 

1  Jour.  Exper.  Med.,  1917,  xxv,  349. 


SALVARSAN   AND   NEOSALVARSAN   IN   TREATMENT   OF   SYPHILIS    853 

strength  of  Schaudinn's  statement  regarding  the  close  biologic  relation- 
ship of  the  spirochetes  of  syphilis  to  trypanosomes,  Uhlenhuth  was  led 
to  employ  atoxyl  in  the  treatment  of  experimental  spirillosis  in  fowls. 
These  experiments,  in  addition  to  those  of  Weisser  and  Metchnikoff, 
demonstrated  beyond  doubt  the  curative  and  prophylactic  properties  of 
atoxyl  in  spirillar  infections.  The  drug  was,  therefore,  administered 
in  cases  of  syphilis  in  the  human  subject.  It  was  found  to  exert  a  very 
beneficial  effect,  especially  in  malignant  forms  of  the  disease.  Further 
experience,  however,  demonstrated  that  atoxyl  was  too  toxic  for  use  in 
the  human  subject,  for  digestive  disturbances,  nephritis,  and  especially 
optic  atrophy  could  be  traced  to  its  use,  even  in  small  doses.  As  a 
therapeutic  agent,  therefore,  it  has  gradually  come  into  disfavor. 

Ehrlich  and  Bertheim  then  made  the  valuable  discovery  that  the 
chemical  constitution  of  atoxyl  was  not,  as  had  been  supposed,  that  of  a 
metarsenate,  but  that  it  was  in  reality  that  of  a  para-amidophenyl- 
arsenate.  Working  on  this  basis,  these  observers  prepared  the  substance 
known  as  arsacetin  (the  sodium  salt  of  acetylpara-amidophenylarsenic 
acid),  which,  although  less  toxic  than  atoxyl,  did  not  fulfil  the  require- 
ments of  a  remedy,  and  its  use  was  discontinued  because  of  its  toxic 
effects  on  human  tissues.  To  lessen  the  relatively  great  toxicity  of  these 
compounds  for  human  tissues  was  a  problem  that  Ehrlich  set  out  to 
solve.  A  most  important  advance  in  this  direction  was  made  when  it 
was  discovered  that  the  unsaturated  triyalent  arsenic  in  arsenobenzol 
and  arsenophenylglycin  has  a  greater  parasiticidal  power  relative  to  its 
toxic  action  on  the  tissues  of  the  host  than  the  pentavalent  arsenic 
compounds,  such  as  atoxyl  and  arsacetin. 

Finally,  after  testing  hundreds  of  these  arsenical  compounds,  it  was 
found  that  in  dioxydiamidoarsenobenzol  we  had  a  substance  that  ap- 
proached the  ideal  in  chemotherapy,  since  it  is  a  drug  that  possesses  a 
maximum  degree  of  parasitotropism  and  a  minimum  degree  of  organo- 
tropism.  Ehrlich  and  Hata  designated  this  light  yellow,  readily  oxidiz- 
able  powder  as  No.  592.  Its  hydrochloric  acid  salt  was  designated  No. 
606,  and  constitutes  the  well-known  salvarsan. 

In  the  published  account  of  their  researches  Ehrlich  and  Hata1 
describe  an  extensive  series  of  investigations  in  experimental  relapsing 
fever,  spirillosis  of  fowls,  and  syphilis  of  rabbits.  These  go  to  prove  the 
high  curative  value  of  salvarsan,  its  relatively  feeble  toxicity  for  the 

iaDie  experimentelle  Therapie  der  Spirillosen,"  Berlin,  1910;  Zeitschr.  f. 
Immunitatsf.,  Ref.  1910,  1123.  Numerous  publications  from  the  Institute  of  Exper- 
imental Therapy,  Frankfort. 


854  CHEMOTHERAPY 

tissues  of  the  infected  animals,  and  its  great  superiority  over  all  other 
substances  that  possess  spirillicidal  properties. 

Clinical  evidence  in  the  treatment  of  human  syphilis  supported  the 
experimental  findings.  On  account  of  the  disadvantages  of  salvarsan, 
due  to  its  insolubility  and  the  necessity  for  using  the  hydrochlorid,  and 
also  in  an  effort  still  further  to  lessen  its  toxicity,  Ehrlich  conducted 
further  researches  to  discover  a  neutral  salt  that  would  be  more  soluble 
and  less  toxic.  As  a  result  of  these  efforts  neosalvarsan,  or  "914,"  has 
been  produced.  This  number  is  significant  of  the  number  of  experiments 
performed  since  the  discovery  of  "592"  and  "606." 

Properties  of  Salvarsan. — Salvarsan  is  the  dihydrochlorid  of  dioxy- 
diamidoarsenobenzol,  and  occurs  as  a  yellow,  crystalline,  hygroscopic 
powder,  very  unstable  in  air,  and  easily  oxidized  to  poisonous  com- 
pounds. It  is  marketed  commercially  put  up  in  small  sealed  vacuum 
tubes.  It  contains  31.57  per  cent,  of  arsenic,  is  readily  soluble  in  water, 
particularly  in  hot  water,  and  yields  a  solution  having  an  acid  reaction. 
If  the  acidity  is  neutralized  by  the  addition  of  caustic  soda  solution, 
the  unsoluble  base  (dioxydiamidoarsenobenzol)  is  precipitated.  If  only 
half  of  this  amount  of  alkali  is  added,  then  the  monohydrochlorid  of 
dioxydiamidoarsenobenzol  is  formed.  If,  in  addition  to  the  amount  of 
caustic  soda  necessary  to  precipitate  the  base,  a  further  quantity  of  al- 
kali is  added,  the  hydrogen  atoms  of  the  phenol  hydroxyls  become  re- 
placed by  Na  and  the  compound  goes  into  solution  as  the  disodium 
salt  of  dioxydiamidoarsenobenzol: 

As  As  As  As 

'S  s\  ^\  ^ 

C1H.NH2|  =  NH2C1H  NH2  =  |NH 


OH  OH  ONa         ONa 

Dihydrochlorid  (aalvarsan)  Disodium  salt  (alkaline  solution) 

Test-tube  experiments  showed  less  spirillicidal  power  of  the  drug 
than  is  demonstrated  in  the  living  animal.  The  following  table  shows 
the  toxicity  of  the  preparation  (Ehrlich  and  Hata) : 


ANIMAL 

METHOD  OP  ADMINISTRATION 

MAXIMUM  TOLERATED  DOSE 

Mouse 

Sub  cutaneous 

1  •  300  per  20  grams 

Mouse.  . 

Intravenous 

1  '  350  per  20  grams 

Rat  

Sub  cutaneous 

0  2    gram  per  kilogram 

Fowl  

Intram  Qgcular 

0  25  gram  per  kilogram 

Fowl  

Intravenous 

0  08  gram  per  kilogram 

Rabbit  

Intravenous 

0.1    gram  per  kilogram 

Rabbit  .  . 

Subcutaneous 

0.15  eram  ner  kiloeram 

SALVARSAN   AND   NEOSALVARSAN   IN   TREATMENT   OF   SYPHILIS     855 

In  experimental  syphilis  of  rabbits  the  minimal  dose  necessary  to 
produce  a  complete  cure  was  found  to  be  between  0.01  and  0.015  gram 
per  kilogram.  The  tolerated  dose  by  intravenous  injection  is  0.1  gram. 
The  curative  dose  of  salvarsan  in  syphilis  of  rabbits  is,  therefore,  only 
from  one-seventh  to  one-tenth  of  the  tolerated  dose. 

Studies  in  the  Toxicity  of  Salvarsan. — During  the  past  two  years 
Dr.  Schamberg,  Dr.  Raiziss,  and  I1  have  studied  the  toxicity  of  salvarsan 
and  have  reached  the  following  conclusions: 

1.  Salvarsan  may  be  used  in  concentrated  solutions  up  to  0.6  gm. 
in  20  c.c.  per  70  kilograms  of  body  weight  in  animals  without  any  evi- 
dent increase  of  toxicity;  in  man,  however,  it  would  appear  that  the 
administration  of  concentrated   solutions  of   salvarsan  increases  the 
toxic  effects. 

2.  The  failure  to  neutralize  solutions  of  salvarsan  with  alkali  leads 
to  an  increase  in  toxicity  of  50  to  60  per  cent,  in  solutions  of  \  to  1  per 
cent,  concentration. 

3.  The  addition  of  a  moderate  excess  of  alkali  beyond  the  amount 
required  for  neutralization  does  not  increase  the  toxicity,  as  determinable 
by  the  duration  of  life  of  the  experimental  animal.    It  is  possible,  how- 
ever, that  it  may  have  other  untoward  effects. 

4.  The  use  of  sterile  fresh  distilled  water  appears  to  possess  advan- 
tages over  sterile  stale  distilled  or  non-distilled  water  as  regards  toxicity, 
although  the  difference  in  our  experiments  was  not  pronounced. 

5.  Salvarsan  in  alkaline  solution  tends  to  undergo  oxidation  on 
standing,  with  consequent  incresed  toxicity,  but  this  substance  and  its 
congeners  vary  considerably  in  the  rapidity  of  oxidation  and  in  the  degree 
of  associated  toxicity.    The  drug  should  be  used  reasonably  promptly 
after  preparation.     If  two  or  three  hours*  delay  is  unavoidable,  the 
solution  should  be  kept  in  a  cylinder  full  to  the  stopper,  so  that  no  air 
is  present. 

6.  Several  different  types  of  reactive  symptoms  may  occur  after 
the  use  of  salvarsan:  (a)  immediate,  (6)  early,  and  (c)  delayed.     The 
immediate  symptoms  are  due  to  a  paresis  of  the  blood-vessels;  the  early 
symptoms  coming  on  a  few  hours  after  the  injection  are  febrile  and 
gastro-intestinal,  and  the  delayed  symptoms  may  be  referable  to  the 
brain  or  the  liver  and  gastro-intestinal  tract.    This  subject  is  discussed 
in  greater  detail  on  page  867. 

7.  Salvarsan  and  its   congeners  are  not   compounds   of  absolute 
chemical  purity.    We  cannot,  therefore,  expect  absolute  constancy  in 
biologic  effects. 

1  Jour.  Cut.  Dis.  Incl.  Syphilis,  April,  1917. 


856  CHEMOTHERAPY 

8.  Salvarsan  and  its  congeners  may  vary,  within  certain  limits,  in 
therapeutic  effect,  and  to  a  greater  degree  in  toxicity.     The  ampules 
obtainable  in  the  open  market  exhibit  striking  variations  in  toxicity. 

9.  Even  the  poorest  compounds,  however,  are  tolerated  by  animals 
in  much  higher  amounts  than  the  maximum  dose  administered  to  man, 
so  that  there  is  nearly  always  a  latitude  of  safety. 

10.  Salvarsan  is  a  safer  substance  than  mercury,  and  can  be  tolerated 
intravenously  by  white  rats  in  fifty  times  the  dose  of  the  latter,  weight 
for  weight. 

Properties  of  Neosalvarsan. — This  is  an  orange-yellow  powder 
possessing  a  peculiar  odor.  It  is  very  unstable  in  the  air  and  is  readily 
soluble  in  water,  yielding  a  yellow  solution  that  is  neutral  to  litmus.  Its 
structure  is  somewhat  more  complex  than  that  of  salvarsan,  being  a 
condensation-product  of  the  latter  and  hydraldit  (formaldehyd  sulph- 
oxylate  of  sodium),  the  reaction  taking  place  according  to  the  follow- 
ing equation: 

As 
/S 

NH2!          1  =  1          I NH2  +  HO.CH/).SO  Na  =  NH 


NH.CH2O.SO  Na+H2O 


While  neosalvarsan  is  less  toxic  than  salvarsan,  and  although  it  is 
much  more  easily  administered  and  largely  free  from  irritative  effects, 
recent  clinical  reports  would  tend  to  show  that  its  spirocheticidal  prop- 
erties are  somewhat  less  than  those  of  salvarsan. 

Sodium  Salvarsan  (Salvarsannatrium). — Ehrlich  has  recently  per- 
fected a  sodium  salt  of  salvarsan  which  may  be  called  a  "neutralized 
salvarsan,"  a  product  regarded  as  possessing  the  curative  value  of  sal- 
varsan and  the  lower  toxicity  and  ease  of  administration  of  neosal- 
varsan. The  reports  of  Wechselman,1  Dreyfus,2  Loeb,3  Fabry  and 
Fischer,4  and  Gutman5  regarding  the  therapeutic  value  of  the  new 

1  Munch,  med.  Wchnschr.,  1915,  Ixii,  177. 

2  Munch,  med.  Wchnschr.,  1915,  Ixii,  178. 

3  Deutsch.  med.  Wchnschr.,  1915,  xli,  338. 

4  Munch,  med.  Wchnschr.,  1915,  Ixii,  612. 

5  Berl.  klin.  Wchnschr.,  1915,  Hi,  No.  16. 


SALVARSAN    AND    NEOSALVARSAN    IN   TREATMENT    OF    SYPHILIS     857 

compound  are  quite  favorable.  It  has  not  been  used  very  extensively 
and  is  not  yet  available  in  America. 

The  doses  are  similar  to  those  of  salvarsan,  averaging  0.4  to  0.6  gm.; 
Wechselman1  has  administered  as  much  as  1  gram  in  a  dose.  The  com- 
pound resembles  salvarsan  in  appearance  except  that  the  powder  has  a 
greenish  hue  and  the  solutions  are  somewhat  darker  in  color.  Solutions 
of  the  drug  oxidize  rapidly  and  must  be  administered  promptly. 

The  drug  is  readily  soluble  in  warm  water  and  does  not  require 
neutralization  with  an  alkali.  It  has  been  administered  in  dilute  and 
concentrated  solutions  dissolved  in  sterile  distilled  water  and  in  the  same 
manner  as  neosalvarsan.  Wechselman  uses  0.4  per  cent,  salt  solution 
in  preparing  the  solution. 

Methods  of  Preparing  Salvarsan  for  Administration. — Soon  after 
the  introduction  of  the  drug  several  methods  of  preparation  and  adminis- 
tration were  suggested.  Many  of  the  disadvantages  attached  to  sal- 
varsan treatment,  and  many  of  the  bad  results  and  complications  re- 
ported, are  to  be  attributed  to  defective  methods  of  preparation  and 
administration  of  the  drug. 

Because  of  the  marked  stability  of  salvarsan  and  neosalvarsan,  the 
following  points  must  be  borne  in  mind : 

1.  The  ampule  containing  the  drug  must  be  intact. 

2.  The  powder  must  be  of  a  yellow  and  not  of  a  gray  or  brownish  color. 
Each  ampule  must  be  carefully  examined  before  the  contents  are 

administered. 

3.  The  drug  should  be  prepared  for  injection  just  prior  to  administra- 
tion. 

It  is  not  necessary  to  describe  the  earlier  methods,  because  these  are 
now  largely  only  of  historic  interest,  and  it  is  quite  generally  accepted, 
from  the  point  of  view  both  of  efficient  treatment  and  of  the  comfort  of 
the  patient,  that  the  intravenous  injection  of  a  dilute  solution  of  the  di- 
sodium  salt  is  the  best  form  of  administration.  For  this  reason  I  shall 
briefly  mention  the  other  methods,  and  describe  the  method  of  intraven- 
ous injection  of  the  alkaline  solution  in  greater  detail  further  on. 

The  Acid  Solution. — When  salvarsan  is  dissolved  in  warm  water  or 
warm  normal  saline  solution,  a  strongly  acid  solution  is  obtained.  In 
this  form  the  drug  is  most  irritating  and  also  most  toxic,  and  when  in- 
jected subcutaneously  and  intramuscularly,  produces  severe  pain  and 
necrosis.  This  method  is  seldom  if  ever  used  at  the  present  time. 

The  Mono-acid  Solution.— If  to  the  watery  acid  solution  half  the 

1  Munch,  med.  Wchnschr.,  1916,  Ixiii,  271. 


858  CHEMOTHERAPY 

amount  of  alkali  necessary  to  produce  complete  neutralization  is  added, 
a  solution  of  the  mono-acid  compound  will  be  formed.  This  solution 
has  been  given  intramuscularly,  but  is  also  extremely  irritating. 

The  Neutral  Suspension. — This  is  the  drug  in  the  form  of  a  precipi- 
tate of  the  base,  prepared  by  adding  to  the  original  acid  solution  just 
sufficient  caustic  soda  to  neutralize  it.  The  method  was  devised  by 
Michaelis  and  Wechselmann,  and  for  some  time  was  the  form  of  adminis- 
tration most  generally  employed  for  subcutaneous  and  intramuscular 
injection.  It  is  probably  less  irritating  than  the  acid  and  clear  alkaline 
solution,  but  occasionally  there  resulted  encapsulation  of  masses  of  ne- 
crotic  tissue  containing  considerable  quantities  of  arsenic. 

Other  Suspensions. — Suspensions  of  salvarsan  in  liquid  paraffin, 
sterile  olive  oil,  oil  of  sesame,  or  almond  oil  are  said  to  keep  for  some 
time  if  placed  in  dark  containers.  This  cannot,  however,  be  always  de- 
pended upon,  and  the  slightest  decomposition  of  the  original  drug  is 
capable  of  producing  marked  toxic  symptoms.  These  suspensions  are 
said  to  be  comparatively  non-irritating,  but  they  may  cause  local  necrosis 
of  tissues,  and  on  account  of  their  slow  absorption,  only  small  amounts 
gain  access  to  the  general  circulation  at  one  time. 

This  method  may,  however,  be  of  value,  especially  in  infants  and  in 
cases  where  slow  absorption  is  desired  in  order  to  prolong  the  effect  of 
the  drug.  Analgesics,  such  as  eucain,  creosote,  or  an  essential  oil,  may  be 
incorporated  in  the  suspension  in  order  to  lessen  the  pain. 

The  suspension  is  so  prepared  that  each  cubic  centimeter  contains 
0.1  gram  of  the  drug. 

Alkaline  Solution  of  the  Disodium  Salt. — This  is  the  form  in  which  the 
drug  should  be  administered  by  intravenous  injection.  It  is  this  solu- 
tion which  Hata  used  in  his  original  experiments,  and  it  was  the  form 
recommended  by  Ehrlich.  I  shall,  therefore,  describe  this  method  in 
detail  a  little  further  on. 


ADMINISTRATION  OF  SALVARSAN  AND  NEOSALVARSAN 

INTRAVENOUS  INJECTION 

Dosage  of  Salvarsan. — Males  receive  in  general  about  0.4  to  0.6  gm. ; 
female  patients  are  usually  given  from  0.25  to  0.5  gm.  In  the  case  of 
weak  and  poorly  nourished  adult  patients  it  in  inadvisable  to  give  more 
than  0.3  or  0.4  gm.  Since  it  has  been  generally  impossible  to  cure  a 
patient  with  a  single  larger  dose,  the  practice  among  syphilologists  at 
present  is  to  give  smaller  doses  frequently  repeated. 


ADMINISTRATION  OF   SALVARSAN   AND    NEOSALVARSAN  859 

For  infants  suffering  from  congenital  syphilis  the  dose  is  from  0.006 
to  0.01  gm.  of  salvarsan  for  every  two  pounds  of  body  weight,  so  that  a 
child  of  eight  pounds  would  receive  from  0.024  to  0.04  gm.  of  salvarsan. 
To  older  children,  weighing  from  40  to  60  pounds,  0.2  to  0.3  gm.  may  be 
given. 

Dosage  of  Neosalvarsan. — This  preparation  is  less  toxic  than  sal- 
varsan, and  may  be  administered  in  larger  doses,  as  from  0.6  to  0.9  gm. 
The  same  general  rule  as  to  the  physical  condition  of  the  patient  should 
apply  here  in  deciding  the  dosage.  At  the  present  time  the  tendency 
is  to  give  adult  patients  about  0.6  gm.  for  three,  four,  and  more  injec- 
tions at  intervals  of  a  week  or  so. 

Frequency  of  Injections;  Intensive  Treatment. — As  was  just  stated, 
there  is  a  distinct  tendency  among  those  of  large  experience  to  regard 
salvarsan  as  a  more  potent  spirocheticid  than  neosalvarsan.  As  pre- 
viously mentioned,  the  original  idea  of  sterilizing  the  patient  with  one 
large  dose  of  the  drug  has  been  largely  abandoned,  especially  in  the 
treatment  of  syphilis  in  any  but  the  earliest  stages.  A  large  number  of 
smaller  doses  are  being  given,  and  the  results  are  controlled  by  the 
Wassermann  reaction  with  blood  and  cerebrospinal  fluid,  in  addition  to 
the  cytologic  changes  in  the  latter.  Thus  from  0.3  to  0.5  gm.  of  sal- 
varsan or  neosalvarsan  is  given  every  week  or  twice  a  week  for  three, 
four,  or  ten  doses  and  more,  depending  upon  the  clinical  results  and  the 
serologic  findings.  In  this  connection  it  must  be  remembered  that  a 
negative  Wassermann  reaction  is  of  little  value  if  blood  has  been  with- 
drawn within  one  week  of  the  last  treatment.  (See  Chapter  XXIII.) 
While  it  is  the  common  practice  to  administer  a  number  of  doses  of  sal- 
varsan or  neosalvarsan,  and  to  follow  this  with  mercury  and  then  with 
salvarsan  again,  this  method  must  be  regarded  as  containing  an  element 
of  danger,  especially  since  Wechselmann  has  drawn  attention  to  the 
fact  that  mercury  acts  as  an  irritant  to  the  kidneys.  It  may  be  stated 
that,  in  general,  most  cases  of  syphilis  require  a  number  of  injections 
of  salvarsan;  this  number  depends  upon  the  age  and  nature  of  the 
infection,  and  should  be  controlled  by  the  Wassermann  reaction  with 
both  blood  and  spinal  fluid. 

Preparation  of  the  Patient. — In  view  of  the  fact  that  many  of  the 
fatalities  due  to  salvarsan  have  been  ascribed  to  defective  kidneys, 
especially  to  kidneys  damaged  by  the  previous  administration  of  mercury, 
it  should  be  a  routine  measure  to  have  the  urine  thoroughly  examined  for 
sugar,  albumin,  and  casts  previous  to  the  administration  of  salvarsan 
or  neosalvarsan. 


860  CHEMOTHERAPY 

While  thousands  upon  thousands  of  injections  have  been  made  with- 
out ensuing  untoward  results,  still  the  administration  of  this  drug  is  not 
without  danger,  and  the  physician  should  familiarize  himself  with  the 
contraindications,  and  subject  his  patient  to  a  careful  physical  examina- 
tion before  undertaking  this  form  of  therapy.  This  subject  will  be 
discussed  further  under  the  head  of  Contraindications  to  Salvarsan 
Therapy. 

In  the  majority  of  cases,  however,  no  contraindications  exist  and  no 
elaborate  preparations  are  necessary.  The  rectum  should  be  emptied 
before  the  injection  is  given,  and  it  is  best  to  administer  the  drug  on  an 
empty  stomach.  After  receiving  salvarsan  the  patient  should  rest  over- 
night under  direct  supervision,  as  in  a  hospital,  the  injection  being  given 
during  the  afternoon  or  early  evening  hours.  The  same  practice  should 
be  followed  with  neosalvarsan,  although  in  thousands  of  instances  pa- 
tients have  received  an  intravenous  injection,  rested  for  an  hour  or  so, 
and  then  returned  to  their  homes. 

Preparation  of  Dilute  Salvarsan  Solution  for  Intravenous  Injection. — 
As  previously  mentioned,  the  physician  should  examine  the  ampule 
containing  the  drug  to  convince  himself  that  it  is  intact.  The  solution 
should  be  prepared  just  before  it  is  injected.  On  account  of  the  oxidation 
that  occurs  it  is  unsafe  to  use  the  drug  if  the  ampule  has  been  open  for 
a  number  of  hours,  and  for  the  same  reason  it  is  not  good  practice  to  pre- 
pare a  bulk  solution  for  a  number  of  patients  unless  the  physician  is  cer- 
tain that  he  will  be  able  to  make  the  injections  quickly  and  without 
interruption. 

The  clear  alkaline  solution  is  most  generally  used.  It  is  prepared 
as  follows : 

1.  The  diluent  should  consist  of  sterile  and  freshly  distilled  water. 
Many  of  the  toxic  and  other  ill  effects  attending  salvarsan  therapy  have 
been  attributed  to  the  use  of  raw  and  stale  water.     Experments  con- 
ducted by  Yakimoff  and  Yakimoff  show  that  the  presence  of  the  endo- 
toxins  of  such  microorganisms  as  Bacillus  coli,  Bacillus  pyocyaneus,  and 
staphylococci  in  the  water  increases  the  toxicity  of  salvarsan  from  two 
to  eight  times,  the  water  alone  and  the  salvarsan  alone  being  without 
effect.    In  office  practice  physicians  may  distil  water  by  means  of  some 
simple  apparatus,  such  as  that  of  Muencke.    The  distilled  water  is  then 
sterilized  in  an  Arnold  sterilizer  for  one-half  hour,  or  by  boiling  for  ten  or 
fifteen  minutes. 

2.  All  glassware  used  should  be  carefully  sterilized,  usually  by  boiling, 
as  in  the  physician's  ordinary  office  sterilizer. 


ADMINISTRATION    OF    SALVARSAN   AND    NEOSALVARSAN  861 

3.  The  water  should  be  hot;  a  good  plan  is  to  place  50  c.c.  in  a  medium- 
sized  (250  c.c.)  Erlenmeyer  flask  and  bring  to  the  boiling-point.  A  mix- 
ing cylinder  may  be  used  instead.  The  ampule  containing  the  drug  is 
wiped  off  with  alcohol,  the  neck  filed  across  and  broken  off,  and  the  con- 
tents gradually  dusted  on  the  surface  of  the  water.  If  the  entire  contents 
of  the  ampule  are  emptied  into  the  water  in  a  bulk,  gelatinous  masses 
may  form  which  dissolve  with  difficulty.  The  solution  is  now  neutralized 
by  adding  a  15  per  cent,  solution  of  caustic  soda  drop  by  drop.  This  neutral- 
ization is  very  important,  as  the  toxicity  of  the  acid  solution  is  extremely  high. 
The  sodium  hydrate  solution  should  be  kept  in  a  non-soluble  glass  con- 
tainer, made  preferably  of  Jena  glass,  in  order  that  no  constituents  of 
the  glass  be  dissolved  with  the  formation  of  a  sediment. 

A  heavy  yellowish  precipitate  is  produced  by  the  addition  of  the 
alkali,  which  clears  up  when  sufficient  alkali  has  been  added;  it  is  un- 
desirable to  add  any  more  alkali  than  is  exactly  necessary  to  clear  the  solu- 
tion, as  an  excess  thereof  increases  the  irritative  effect  of  the  preparation. 
(If  too  much  alkali  has  been  inadvertently  added,  the  excess  can  be 
neutralized  by  the  addition  of  a  few  drops  of  a  10  per  cent,  solution  of 
chemically  pure  hydrochloric  acid.  If  the  acid  produces  a  cloud,  this 
can  again  be  cleared  by  a  drop  or  two  of  alkali.)  When  doubt  exists 
concerning  the  reaction  of  the  solution,  red  and  blue  litmus-paper  should 
be  employed.  The  amount  of  alkali  necessary  is  about  4  drops  of  a 
15  per  cent,  solution  for  each  0.1  gm.  of  salvarsan;  thus,  for  0.6  gin., 
1.14  c.c.,  or  about  from  23  to  45  drops  of  15  per  cent,  solution  of  caustic 
soda,  would  be  required.  Citron  gives  the  following  table: 

0.2  gm.  salvarsan  requires  0.38  c.c.  of  15  per  cent,  sodium  hydroxid  =  8  drops. 
0.3  gm.  salvarsan  requires  0.54  c.c.  of  15  per  cent,  sodium  hydroxid  =  12  drops. 
0.4  gm.  salvarsan  requires  0.76  c.c.  of  15  per  cent,  sodium  hydroxid  =  15  or  16  drops. 
0.5  gm.  salvarsan  requires  0.95  c.c.  of  15  per  cent,  sodium  hydroxid  =  19  or  20  drops. 
0.6  gm.  salvarsan  requires  1.14  c.c.  of  15  per  cent,  sodium  hydroxid  =  23  to  24  drops. 

A  drop  more  of  the  alkali  than  is  just  necessary  to  produce  the  clear 
solution  should  be  added.  If  this  is  not  done,  on  cooling  the  solution 
may  show  a  precipitate;  this  can  be  redissolved  by  the  addition  of  a 
drop  of  alkali.  When  ready  for  use  the  solution  should  be  slightly  alka- 
line. When  the  solution  is  neutralized,  water  should  be  added  to  increase 
the  quantity  to  200  c.c.  for  0.6  gram  of  the  drug  or  to  150  c.c.  for  0.4, 
although  the  drug  if  injected  slowly  may  be  given  in  30  to  50  c.c. 

I  do  not  believe  that  any  advantage  is  gained  by  the  employment 
of  salt  solution  instead  of  distilled  water.  If  this  is  preferred,  the  sal- 
varsan should  be  first  dissolved  in  distilled  water,  neutralized,  and  then 
a  sterile  0.5  per  cent,  solution  of  chemically  pure  sodium  chlorid  added 
to  make  up  the  desired  volume. 


862 


CHEMOTHERAPY 


The  temperature  of  the  solution  at  the  time  of  injection  should  be 
about  that  of  the  body. 

This  flask  is  thoroughly  shaken  to  insure  an  even  diffusion  of  the 
drug,  and  the  contents  poured  into  the  cylinder  from  which  it  is  admin- 
istered, being  filtered  into  this  cylinder  through  a  piece  of  sterile  gauze 
in  order  to  remove  any  bits  of  broken  glass  from  the  ampule  that  may 
have  gained  access  or  any  other  insoluble  particles.  (See  Fig.  146.) 


FIG.  146. — INTRAVENOUS  INJECTION  OF  SALVARSAN. 

With  this  apparatus  the  operator  may  give  an  injection  without  assistance. 
Notice  the  three-way  cock,  which  permits  the  flow  of  salt  solution  or  salvarsan  solu- 
tion at  will.  Usually  a  tourniquet  composed  of  a  simple  rubber  tubing  and  held  in 
position  by  a  hemostat  is  better  than  the  one  shown,  as  the  operator  can  quickly  re- 
lease it  with  least  disturbance  and  loss  of  time.  Note  the  funnels  in  both  con- 
tainers for  straining  the  salvarsan  solution  and  distilled  water  or  salt  solution.  (After 
the  apparatus  of  Boehm.) 

Preparation  of  Dilute  Neosalvarsan  Solution  for  Intravenous  Injec- 
tion.— This  drug  is  readily  soluble  in  water,  forming  a  clear  solution 
which  is  ready  for  use.  File  the  neck  of  the  ampule,  cleanse  it  with 
alcohol,  and  break  open.  The  contents  are  emptied  directly  into  a  flask 
containing  100  to  150  c.c.  of  warm,  sterile,  freshly  distilled  water.  On 
gentle  agitation  the  drug  rapidly  dissolves.  Hot  water  should  not  be 
used,  nor  should  a  solution  be  heated  once  it  has  been  made.  From 
the  mixing  flasks  the  solution  is  poured  into  the  cylinder  through  a 


ADMINISTRATION   OF   SALVARSAN   AND   NEOSALVARSAN 


863 


piece  of  sterile  gauze,  which  filters  out  any  bits  of  glass  or  other  insoluble 
particles.  For  administration  any  simple  apparatus,  such  as  that  shown 
in  Fig.  147,  answers  all  purposes. 

Injecting  Apparatus. — A  number  of  different  apparatus  for  the  ad- 
ministration of  dilute  solutions  of  salvarsan  and  neosalvarsan  have  been 
invented  and  marketed.  That  shown  in  Fig.  146  is  well  adapted  for  the 
purpose;  one  cylinder  carries  sterile  normal  salt  solution  and  the  second 


FIG.  147. — METHOD  OF  MAKING  INTRAVENOUS  INJECTION  BY  GRAVITY. 
This  method  is  suitable  for  the  intravenous  administration  of  salvarsan  or  anti- 
streptococcus  serum,  etc.  The  needle  has  been  entered  into  a  prominent  vein 
(indicated  by  a  flow  of  blood);  the  tubing  has  been  attached  by  means  of  a  metal 
tip  which  fits  the  needle  easily  and  snugly;  the  tourniquet  has  been  loosened  and  the 
injection  is  being  given. 


holds  the  salvarsan  or  neosalvarsan  solution.  By  fastening  the  cylinders 
to  an  upright  rod  attached  to  the  side  of  the  table  their  height  may  be  ad- 
justed as  desired,  and  by  means  of  the  special  cock  the  operator  may 
insert  the  needle  and  allow  the  salt  solution  to  flow  in  until  he  is  sure  of 
having  satisfactorily  penetrated  the  vein,  when  he  may  turn  on  the  sal- 
varsan solution.  In  this  manner  the  physician  is  enabled  to  give  an 
injection  without  assistance. 

Or,  if  desired,  the  simpler  apparatus  shown  in  Fig.  147  may  be  used. 


864  CHEMOTHERAPY 

This  consists  of  a  single  cylinder  to  the  narrow  lower  end  of  which  about 
five  feet  of  rubber  tubing  are  attached.  A  piece  of  glass  tubing  is  in- 
serted at  the  lower  end  to  serve  as  a  window,  and  at  the  end  there  is  an 
arrangement  whereby  it  can  easily  be  attached  to  the  needle  used  for 
making  venipuncture.  A  clamp  is  placed  on  the  tubing  at  some  con- 
venient place,  where  it  may  be  operated  by  an  assistant. 

Whatever  apparatus  is  employed,  it  should  be  sterilized  before  use. 
The  cylinders  and  needle  are  boiled  in  an  office  sterilizer,  and  the  tubing 
is  cleansed  by  running  sterile  salt  solution  or  water  through  it.  The 
needle  should  have  a  sharp  point  with  a  short  beveled  edge,  as  a  long- 
pointed  needle  may  pierce  the  vein  through  and  through.  Sufficient 
sterile  distilled  water  or  normal  salt  solution  is  added  to  fill  the  rubber 
tubing,  while  the  cylinder  should  contain  an  additional  10  or  15  c.c. 
If  the  double  cylinders  are  used  (Fig.  146),  one  should  contain  from  20  to 
30  c.c.  of  salt  solution  or  water  and  the  second  the  solution  of  the  drug. 
The  solution  of  drug  is  then  filtered  into  the  second  cylinder,  and  the 
pinch  cock  opened  for  a  minute  to  be  sure  that  all  air  has  been  expelled. 

2.  The  patient  should  lie  on  a  bed  or  on  an  operating  table.     An  arm 
— usually  the  patient's  left  in  the  case  of  right-handed  operators — is 
prepared  by  placing  a  few  towels  around  it  and  a  firm  tourniquet  is  ap- 
plied above  the  elbow.     Whatever  material  is  used,  whether  rubber  or  a 
broad  muslin  bandage,  it  should  be  fastened  with  a  hemostat,  for  when 
it  becomes  necessary  to  unfasten  it,  this  may  be  done  quickly  and  with 
least  disturbance  by  the  operator,  who  simply  unfastens  the  hemostat. 
The  skin  over  a  prominent  vein  may  be  cleansed  with  green  soap  and 
water,  followed  by  alcohol  and  1  : 100  bichlorid  solution,  or  simply  by 
adding  one  or  two  coats  of  10  per  cent,  tincture  of  iodin,  which  is  washed 
off  with  cotton  and  alcohol  just  before  the  needle  is  inserted.     In  fat 
subjects  a  vein  can  frequently  be  felt  when  it  cannot  be  seen.     Occasion- 
ally it  may  be  necessary  to  infiltrate  the  skin  with  sterile  eucain  solution 
and  expose  the  vein  by  incision.    In  nervous  subjects  the  routine  opera- 
tion of  inserting  the  needle  can  be  made  practically  painless  by  infil- 
trating the  skin  over  a  vein  with  a  few  drops  of  a  sterile  1  per  cent, 
solution  of  eucain. 

3.  The  operator  now  passes  the  needle  into  a  vein.    With  experience, 
considerable  skill  is  gained  in  performing  this  little  operation.    A  free 
flow  of  blood  indicates  that  the  needle  has  been  properly  inserted,  and 
one  can  easily  tell  by  the  sense  of  touch  whether  the  needle  is  free  in  the 
lumen.    The  tourniquet  is  then  quickly  and  deftly  released  by  the  oper- 
ator, the  clamp  on  the  tube  is  opened,  and  while  the  blood  is  flowing  from 


ADMINISTRATION   OF   SALVARSAN   AND   NEOSALVARSAN  865 

the  vein  and  the  saline  solution  or  water  from  the  tubing  the  nozzle  of 
the  latter  is  fitted  into  the  needle  quickly  and  securely  while  the  latter 
is  held  firmly  in  the  vein.  The  appearance  of  bulging  at  the  side  of  the 
vein  and  the  occurrence  of  pain  indicate  that  the  needle  has  not  been 
properly  inserted.  In  this  event  the  clamp  should  be  fastened,  the 
needle  withdrawn,  the  tourniquet  adjusted,  and  another  vein  punctured. 
It  is  useless  to  attempt  to  enter  the  same  vein  at  the  same  point. 

4.  The  introduction  of  a  full  dose  of  salvarsan  or  neosalvarsan  will 
usually  take  from  ten  to  twenty  minutes  or  thereabouts.     If  the  flow  is 
retarded,  the  needle  may  be  turned  gently  and  slightly  so  as  to  change 
the  relation  of  the  bevel  to  the  wall  of  the  vein. 

5.  When  salt  solution  forms  the  first  portion  of  the  injection,  no 
harm  has  been  done  if  perivascular  infiltration  occurs.     This  method 
gives   added  assurance  to  the  operator;    indeed,  it  should  never  be 
omitted  when  salvarsan  is  being  injected,  and  it  is  a  good  general  rule  to 
have  the  first  portion  of  the  injection  consist  of  normal  salt  solution. 

6.  After  the  requisite  dose  has  been  injected,  a  few  cubic  centimeters 
of  salt  solution  are  again  permitted  to  flow  into  the  vein,  so  that  the  tub- 
ing and  needle  are  washed  free  from  salvarsan,  and  at  no  time  does  the 
drug  come  in  contact  with  the  tissues. 

7.  The  needle  is  then  quickly  withdrawn,  and  the  site  of  the  punc- 
ture sealed  with  collodion  and  cotton  after  the  iodin  has  been  removed 

by  washing  with  alcohol. 

Intravenous  Administration  of  Salvarsan  in  Concentrated  Solution. — 
While  animal  expermients  have  shown  us  that  concentrated  solutions 
of  salvarsan  are  not  markedly  toxic,  the  administration  of  concentrated 
solutions  to  persons  appears  to  increase  the  chances  of  disagreeable 
after-effects.  This  is  particularly  true  if  the  concentrated  solution  is 
injected  quickly,  which  is  the  natural  tendency  of  the  physician  when 
all  is  working  smoothly. 

In  preparing  a  concentrated  solution,  20  c.c.  of  hot,  sterile,  freshly 
distilled  water  are  placed  in  a  small  Erlemneyer  flask  or  cylinder  and  the 
drug  added  in  small  amounts  followed  by  brief  shaking,  until  all  has 
been  added  and  dissolved.  The  clear  solution  is  now  neutralized  by  the 
addition  of  a  15  per  cent,  solution  of  caustic  soda  in  exactly  the  same  manner 
as  in  the  preparation  of  the  dilute  solution.  The  physician  must  never 
omit  this  step',  the  intravenous  injection  of  a  concentrated  and  acid  solution 
of  salvarsan  is  most  dangerous  and  has  resulted  in  fatalities. 

The  solution,  which  should  be  filtered  through  sterile  gauze  or  paper, 

is  now  taken  up  into  a  sterile  20-  or  30-c.c.  syringe  and  air  dispelled; 
55 


866  CHEMOTHERAPY 

the  Record,  Luer,  and  Burroughs- Wellcome  syringes  are  recommended. 
The  arm  of  the  patient  is  prepared  as  described  above  and  a  tourniquet 
applied,  which  may  be  quickly  released  by  the  operator  with  least  dis- 
turbance. The  needle  is  now  passed  into  a  vein,  and  after  a  free  flow  of 
blood  appears  the  syringe  is  gently  adjusted  and  the  injection  slowly 
made  (at  least  one  or  two  minutes  should  be  required) .  Great  care  should 
be  exercised  that  none  of  the  solution  enters  the  tissues.  The  syringe 
shown  in  Fig.  139  is  particularly  well  adapted  for  this  work,  as  it  enables 
the  physician  to  puncture  the  vein  with  the  syringe  containing  a  small 
amount  of  salt  solution,  and  also  enables  him  to  wash  in  a  small  amount 
of  the  same  at  the  close  of  the  operation  and  thus  avoid  injecting  salvar- 
san  into  the  tissues. 

Intravenous  Administration  of  Neosalvarsan  in  Concentrated  So- 
lution.— Neosalvarsan,  being  less  toxic  than  salvarsan,  may  be  readily 
administered  in  concentrated  solution.  The  dose  of  drug  to  be  admin- 
istered is  dissolved  in  20  c.c.  of  warm,  sterile,  freshly  distilled  water  in  a 
small  cylinder  and  requires  no  neutralization.  The  clear  solution  is 
now  taken  up  into  a  sterile  syringe  and  injected  in  the  manner  described 
above. 

After-care  of  the  Patient. — In  the  majority  of  instances  the  adminis- 
tration of  salvarsan  is  not  followed  by  unpleasant  symptoms.  This 
depends,  however,  to  a  considerable  extent  upon  the  nervous  constitu- 
tion of  the  patient.  Many  persons  will  complain  of  a  feeling  of  fullness 
and  may  perspire  freely  for  a  short  time.  There  may  be  slight  pain  at 
the  site  of  injection  and  in  the  axilla  of  the  injected  side.  As  was  pre- 
viously mentioned,  salvarsan  should  be  administered  at  the  patient's 
home  or  in  a  hospital,  followed  by  rest  in  bed  until  the  next  morning. 
When  neosalvarsan  is  injected,  robust  persons  may,  after  resting  for  an 
hour  or  so,  travel  homeward.  Occasionally  severer  reactions  follow 
salvarsan  administration,  and  these  may  be  considered  under  the  head 
of  after-effects. 

After-effects  of  Salvarsan.— Within  an  hour  or  two  after  its  admin- 
istration arsenic  is  excreted  by  the  kidneys  and  bowels  and  nausea  may  be 
complained  of.  If  catarrh  of  the  stomach  is  present,  severe  vomiting  may 
ensue.  The  nausea  is  relieved  by  sipping  a  little  hot  water;  hot  applica- 
tions over  the  stomach  and  small  amounts  of  carbonated  water  or  cham- 
pagne will  usually  control  the  vomiting.  It  may  occasionally  be  neces- 
sary to  administer  y%  grain  of  morphin  hypodermically,  especially  if  the 
patient  is  of  a  neurotic  temperament.  Headache  may  occasionally  de- 
velop, and  is  due  to  a  neurotic  condition,  constipation,  anxiety,  hunger, 


ADMINISTRATION    OF    SALVARSAN    AND    NEOSALVARSAN  867 

or  possibly  to  an  increase  in  the  exudate  of  the  syphilitic  process.  Diar- 
rhea may  be  observed  in  a  few  cases;  this  is  readily  controlled  by  the 
administration  of  bismuth.  Chills  and  fever  are  more  infrequent  at  the 
present  time,  the  result  probably  of  using  freshly  distilled  water  and 
somewhat  smaller  doses  of  the  drug.  In  some  cases  an  arsenic  rash, 
in  the  form  of  an  erythema,  may  follow  injection.  This  is  not  the 
Jarisch-Herxheimer  reaction,  which  is  found  only  in  syphilis,  the  ar- 
senic rash  having  been  observed  in  non-syphilitic  persons  injected  with 
the  drug.  There  is  no  evidence  to  show  that  salvarsan  or  neosalvarsan 
will  injure  healthy  kidneys,  although  a  mild  transient  albuminuria  may 
follow  the  injections  in  some  instances.  When,  however,  the  kidneys 
have  been  damaged  by  mercury,  salvarsan  may  give  rise  to  an  acute  irri- 
tation, and,  indeed,  Wechselmann  ascribes  many  of  the  salvarsan  casual- 
ties to  this  condition,  and  issues  the  warning  that,  while  mercury  may 
follow  salvarsan,  it  should  never  precede  it.  The  good  general  effects 
following  the  administration  of  salvarsan  are  manifested,  as  a  rule,  in  a 
sense  of  well-being,  and  not  infrequently  patients  who  are  anemic, 
poorly  nourished,  and  despondent  in  a  short  time  become  healthy,  active, 
and  cheerful.  This  may  be  due  in  part  to  a  psychic  effect,  but  there 
is  frequently  evidence  of  a  far-reaching  change  in  the  nutrition  of  the 
patient. 

Reactive  Manifestations  After  the  Use  of  Salvarsan. — The  various 
reactive  symptoms  which  not  uncommonly  follow  the  administration 
of  salvarsan  or  its  congeners,  have  been  assigned  by  different  clinical 
observers  to  widely  diverse  causes.  The  generally  accepted  views  have 
undergone  considerable  modification  from  time  to  time.  At  the  present 
day  our  knowledge  of  the  subject  is  still  imperfect  and  unsatisfactory. 
The  hypotheses  which  have  been  advanced  but  inadequately  explain 
the  phenomena.  The  subject  is  particularly  complicated  because  the 
physiologic  and  toxicologic  effects  of  a  relatively  new  and  extremely 
complex  compound  are  not  accurately  known.  Dr.  Schamberg,  Dr. 
Raiziss,  and  I1  have  devoted  considerable  time  to  experimental  studies 
bearing  upon  the  causative  factors  involved  in  the  production  of  salvar- 
san reactions,  and  the  following  summarizes  our  results  and  views  upon 
this  subject: 

In  order  that  the  phenomena  developing  after  the  administration 
of  salvarsan  may  be  better  discussed  we  have  thought  it  wise  to  classify 
the  symptoms  into  three  groups:  (a)  the  immediate  symptoms,  (6)  the 
early  symptoms,  and  (c)  the  late  symptoms. 

1  Jour.  Cutan.  Dis.  inch  Syph.,  April,  1917. 


868  CHEMOTHERAPY 

(a)  The  Immediate  Symptoms.— These  phenomena  are  observed 
during  the  intravenous  administration  of  the  drug  or  within  a  few  min- 
utes after  the  completion  of  the  same.  At  times  patients  during  in- 
fusion of  the  solution  will  state  that  they  taste  the  drug.  Inquiry  will 
usually  elicit  the  reply  that  the  taste  is  like  that  of  ether.  A  burning 
sensation  of  the  tongue  or  lips  may  be  complained  of;  these  are  usually 
the  precursor  of  a  train  of  other  symptoms.  The  first  objective  evidence 
of  immediate  reaction  is  flushing  of  the  face;  this  may  be  slight  and 
transitory,  or  may  be  pronounced  and  accompanied  by  injection  of  the 
conjunctive,  lacrimation,  edema  and  swelling  of  the  lips,  tongue,  and 
eyelids,  an  anxious  expression  of  the  countenance,  nausea  followed 
by  vomiting  and  retching,  and  this,  in  turn,  by  profuse  perspiration. 
In  some  cases  cough,  respiratory  embarrassment,  and  dyspnea  are 
observed.  The  pulse  at  first  is  full  and  bounding,  but  later  slow  and  of 
very  small  volume,  in  which  event  it  is  usually  accompanied  by  a  pro- 
nounced pallor.  In  severe  cases  the  patient  may  lose  consciousness  and 
the  pulse  may  be  scarcely  palpable.  In  rare  instances  death  has  been 
reported.  An  urticarial  eruption  appearing  during  the  congestive  stage 
is  one  of  the  more  uncommon  manifestations,  as  is  also  a  severe  pain  in 
the  lumbar  region.  These  symptoms  may  disappear  within  fifteen  to 
thirty  minutes,  and  be  followed  by  no  other  phenomena,  or,  as  more 
commonly  happens,  other  symptoms,  as  chills,  develop  within  a  brief 
period  or  several  hours  later.  This  secondary  complex  we  prefer  to 
discuss  as  group  b,  as  these  symptoms  not  infrequently  appear  several 
hours  after  the  injection  of  salvarsan,  even  when  they  are  not  preceded 
by  the  vasomotor  phenomena  just  described. 

(6)  The  Early  Symptoms. — These  symptoms  consist  of  chilliness  or  a 
distinct  rigor,  headache,  vertigo,  nausea,  vomiting,  diarrhea,  and  rise 
of  temperature,  usually  100°  to  102°  F.  But  few  of  these  may  be  present 
and  they  may  be  so  mild  as  to  merely  make  the  patient  feel  "queer,"  or 
there  may  be  repeated  chills,  numerous  attacks  of  emesis,  and  profuse 
and  protracted  diarrhea.  Occasionally  severe  pains  in  the  legs  and  back 
are  complained  of.  This  group  of  symptoms  usually  passes  off  in  twelve 
to  twenty-four  hours,  and  is  followed  by  a  feeling  of  lassitude  or  weakness. 
More  uncommonly  vomiting  and  diarrhea,  associated  with  some  ele- 
vation of  temperature,  may  continue  for  a  number  of  days,  the  patient 
during  this  period  being  unable  to  retain  any  nourishment.  In  some 
cases  the  urine  may  be  scanty  and  contain  albumin  and  casts. 

Various  eruptions  have  been  observed  at  times  after  the  adminis- 
tration of  salvarsan,  appearing  either  in  a  few  hours  or  after  several 


ADMINISTRATION    OF    SALVARSAN    AND    NEOSALVARSAN  869 

days.  The  most  common  are  urticarial  and  scarlatinoid  and  morbilli- 
form  erythemas,  and  in  rare  cases  purpura.  In  some  instances  an  itching 
of  the  skin  or  pruritus  without  accompanying  eruption  has  been  noted. 
Most  of  these  eruptions  are  ephemeral  and  disappear  in  a  day  or  two. 
Later  and  more  persistent  eruptions  have  been  observed  to  occur  from 
six  to  ten  days  after  the  administration  of  the  drug.  These  late  erup- 
tions are  more  common  after  intramuscular  injection.  Some  instances 
of  universal  exfoliating  dermatitis  have  been  reported,  persisting  for 
weeks,  with  fever  and  debility,  and  sometimes  leading  to  a  fatal  termi- 
nation. 

(c)  The  Late  Symptoms. — Delayed  reactions  may  come  on  after 
twenty-four  hours,  in  which  event  they  usually  consist  of  vomiting, 
fever,  and  diarrhea,  similar  to  the  intermediate  reactions.  More  rarely 
serious  and  even  fatal  reactions  may  develop  about  three  days  after  the 
administration  of  the  drug.  In  these  cases  the  phenomena  are  either 
referable  to  the  brain  or  the  liver.  In  the  severe  cases  there  may  be 
headache,  vomiting,  muscular  twitchings,  epileptiform  convulsions,  di- 
lation of  the  pupils,  absent  reflexes,  coma,  and  death.  These  symptoms 
are  usually  the  expression  of  edema  of  the  brain  or  of  encephalitis  hem- 
orrhagica.  Meirowsky  and  Kretzmer,1  in  their  splendid  monograph  on 
the  salvarsan  therapy  of  syphilis,  present  a  critical  analysis  of  the  fatal- 
ities following  the  use  of  salvarsan.  Of  109  fatal  terminations  recorded  in 
literature,  41  per  cent,  occurred  in  the  secondary  period  and  27  per  cent, 
in  late  nerve  syphilis.  Three-fifths  of  the  fatalities  during  the  secondary 
stage  resulted  from  encephalitis.  Forty-five  per  cent,  of  these  occurred 
in  cases  in  which  the  dose  exceeded  0.5  gm.  Grouping  all  of  the  cases 
of  single  and  multiple  injections,  66  per  cent,  of  the  cases  received 
doses  over  0.5  gm.  The  size  of  the  dose  is,  therefore,  a  most  important 
factor  in  the  causation  of  encephalitis.  The  danger  decreases  in  pro- 
portion to  the  duration  of  the  interval  between  the  injections. 

Another  rare  syndrome  following  the  administration  of  salvarsan  is 
characterized  by  severe  jaundice  accompanied,  as  a  rule,  by  fever.  This 
may  appear  in  from  three  days  to  several  weeks  after  treatment.  Such 
accidents  are  much  more  frequent  after  intramuscular  than  after  intra- 
venous injections.  Most  cases  of  postsalvarsan  jaundice  pursue  a 
favorable  course,  but  there  are  exceptional  cases  which  terminate  fatally 
with  the  symptoms  and  autopsy  findings  of  acute  yellow  atrophy  of 
the  liver. 

The  Causes  of  Reaction. — At  the  outset  it  should  be  clearly  stated 

1  Prakt.  Ergeb.  auf  dem  debist  der  Haut  und  Geschlechtk,  Wiesbaden,  1914. 


870  CHEMOTHERAPY 

that  there  is  no  single  cause  that  will  explain  the  varied  reactive  phe- 
nomena following  the  administration  of  dioxydiamino-arsenobenzol. 
Indeed,  it  is  possible  that  the  various  groups  of  symptoms  may  be  due 
to  different  causes. 

It  will  perhaps  lead  to  greater  clarity  if  we  classify  the  causes  of 
reaction  under  three  heads:  (1)  Factors  related  to  the  patient;  (2) 
factors  related  to  the  technic  of  administration,  and  (3)  factors  related 
to  the  chemical  compound  employed. 

1.  Factors  Related  to  the  Patient. — There  can  be  no  doubt  that  indi- 
vidual susceptibility  plays  a  part  in  reactions.     Patients  suffering  from 
syphilis  vary  greatly  in  their  physical  condition  in  the  degree  of  func- 
tional and  organic  integrity  of  the  various  organs.    As  a  class,  too,  they 
are  prone  to  be  neurasthenic,  and  the  mental  state  at  the  time  of  injection 
doubtless  influences  the  incidence  of  some  of  the  less  reactive  phenom- 
ena.   Patients  may  vary  in  their  psychic  response  to  the  operation 
per  se,  in  the  manner  in  which  they  react  to  errors  of  technic,  and  their 
susceptibility  to  the  drug  itself.    Two  individuals  may  receive  a  solution 
of  the  same  drug  prepared  at  the  same  time,  and  one  may  suffer  reaction 
and  the  other  remain  free.    One  of  the  writers  had  a  striking  example 
of  this  in  his  practice.    A  physician  and  his  patient  each  received  half 
of  the  quantity  of  an  alkaline  solution  of  salvarsan  from  the  same  mixing 
cylinder.      The    physician    shortly    afterward    developed    pronounced 
chills,  elevation  of  temperature  to  102°  F.,  and  severe  pains  in  the  legs; 
the  other  patient,  contrary  to  instructions,  ate  a  hearty  meal  an  hour 
later  and  took  a  two  hours'  train  ride  to  a  neighboring  city,  but  suffered 
no  reaction.    There  can  be  no  doubt,  therefore,  that  the  personal  element 
may  be  a  factor  in  causing  or  influencing  reactive  symptoms,  but  it  is, 
in  our  opinion,  not  the  dominant  factor. 

2.  Factors  Relating  to  the  Technic. — These  factors  have  been  given 
much  attention  in  literature,  and  different  observers  have  attributed 
great  significance  to  one  or  another  error  of  technic.    The   greatest 
interest  and  controversy  have  attached  to  the  "water  error"  or  "Wasser 
fehler"  of  Wechselmann.    Wechselmann  advanced  the  hypothesis  that 
water  impurities,  chiefly  bacterial  proteins,  were  largely  responsible  for 
the  febrile  and  gastro-intestinal  reactions.     This  view  was  accepted 
and  indorsed  by  Ehrlich,  Max  Miiller,  and  many  others.     Yakimoff 
and  Yakimoff  showed  that  the  presence  of  bacteria  in  the  salvarsan 
solution  increased  the  toxicity  to  a  varying  degree,  depending  upon  the 
type  of  organisms  present.     The  colon  bacillus  increased  the  toxicity 
most,  the  Bacillus  pyocyaneus  less,  and  other  bacteria  little  or  not  at  all. 


ADMINISTRATION    OF    SALVARSAN    AND    NEOSALVARSAN  871 

Gemerich  believed  that  organic  contamination  of  the  water  by  sapro- 
phytes was  the  cause  of  a  number  of  unfortunate  consequences.  He 
believed  that  certain  poisonous  substances  in  the  bacteria  are  not  de- 
stroyed by  boiling,  and  that  they  may  produce  febrile  reaction  in  the 
patient.  Fresh  distilled  water  that  did  not  contain  bacterial  bodies 
was  less  apt  to  produce  fever. 

Arzt  and  Kerl,  Nobel  and  Peller,  Wachenfeld,  and  others  do  not  admit 
the  "Wasser  fehler"  as  the  cause  of  fever.  Luitheln  and  Mucha  also 
dissent  from  this  view,  and  regard  the  fever  as  due  to  a  resorption  of 
broken-down  cell  products;  headache,  vertigo,  and  vomiting  are  also 
regarded  by  them  as  due  to  this  cause.  Neisser  had  previously,  in  1910, 
advanced  the  view  that  the  febrile  symptoms  were  due  to  the  rapid 
destruction  of  innumerable  spirochetes  and  the  setting  free  of  endo- 
toxins  which  reached  the  blood-stream  and  induced  the  symptoms. 

Emery  believed  that  contamination  of  the  water  with  copper,  leas, 
or  silicates  from  the  distilling  apparatus,  increased  the  toxicity  of  sal- 
varsan  and  caused  reactive  manifestations. 

Gonder,  at  the  request  of  Ehrlich,  carried  out  on  animals  some  experi- 
ments on  the  toxicity  of  salvarsan  solutions  to  which  small  quantities 
of  calcium  and  magnesium  salt,  which  are  commonly  found  in  tap-water, 
were  added.  He  found  that  these  increased  the  toxicity  of  the  drug. 

Matzenauer  believed  that  alkali  which  was  dissolved  out  of  the  glass 
container  was  a  factor  in  reactions. 

Dreyfus  counseled  doubly  distilling  the  water  and  boiling  the  in- 
fusion apparatus  in  distilled  water  and  not  in  ordinary  water. 

From  this  mass  of  divergent  opinions  it  is  difficult  to  draw  any  clear 
and  definite  deductions.  While  the  "water  error"  is  a  factor  in  reactions, 
it  is  probably  much  less  constantly  responsible  than  has  been  alleged 
by  Wechselmann  and  others.  Many  of  the  factors  referred  to  are  doubt- 
less capable  of  causing  fever  and  gastro-intestinal  symptoms  in  certain 
patients,  but  no  one  of  these  causes  is  responsible  for  the  majority  of 
the  reactions.  There  are  other  faults  of  technic  which  may  likewise 
be  responsible  for  reactions,  such  as  the  use  of  too  hot  or  too  cold  water, 
too  acid  or  too  alkaline  solutions,  the  employment  of  too  small  or  too 
great  a  volume  of  water,  the  too  rapid  infusion  of  the  solution,  etc. 
Then,  too,  the  improper  preparation  of  the  patient  for  the  injection  or 
improper  after-treatment.  The  intravenous  administration  of  the  drug 
shortly  after  the  eating  of  a  meal  or  the  partaking  of  food  too  soon  after 
the  injection  may  induce  gastro-intestinal  reactions.  Physical  over- 
exertion  immediately  before  or  directly  after  treatment  may  likewise 
be  responsible  for  reactive  phenomena. 


872  CHEMOTHERAPY 

3.  Factors  Related  to  the  Medicament. — We  are  strongly  inclined  to 
believe  that  in  the  interpretation  of  the  causes  of  reactions  following 
the  administration  of  salvarsan  (and  we  are  employing  this  term  to 
indicate  dioxydiamino-arsenobenzol,  irrespective  of  the  trade  name 
under  which  it  is  marketed),  too  great  a  stress  has  been  laid  upon  errors 
of  technic  and  upon  personal  factors,  and  too  little  attention  has  been 
paid  to  a  study  of  causes  related  to  the  compound  itself.  In  order  to 
intelligently  discuss  this  aspect  of  the  question  it  is  necessary  to  refer 
to  the  chemistry  of  the  substance  under  consideration.  Salvarsan  is  an 
extremely  complex  substance,  perhaps  the  most  complex  drug  employed 
in  medicine.  Before  its  final  elaboration  numerous  intermediate  products 
must  be  prepared.  The  purity  of  the  final  compound  will  depend  to  a 
certain  degree  upon  the  character  of  some  of  the  precedent  intermediate 
substances.  Even  if  these  be  in  a  pure  state,  certain  impurities  are  apt 
to  develop  during  the  process  of  reduction  with  sodium  hydrosulphite. 
Salvarsan  is  precipitated  by  ether  out  of  a  hydrochloric  acid  methyl 
alcohol  solution  of  the  base;  other  substances  in  minute  quantities  may 
be  precipitated  in  addition  to  salvarsan.  Inasmuch  as  salvarsan  cannot 
be  purified  by  repeated  crystallization,  we  cannot  regard  salvarsan  as  an 
absolutely  pure  compound.  A  number  of  analyses  made  by  us  corrob- 
orate this  statement.  Salvarsan  does  not  give  arsenic  values  quite  up 
to  the  theoretic  amount,  and,  furthermore,  there  are  elements  present 
which  have  no  place  in  the  chemical  formula.  We  have  found  in  our 
analyses  that  salvarsan  contains  on  an  average  1  to  2  per  cent,  of 
sulphur;  our  own  product,  arsenobenzol,  likewise  contains  sulphur.  The 
sulphur  doubtless  becomes  attached  to  the  arsenic  during  the  process 
of  reduction. 

Inasmuch  as  salvarsan  is  not  an  absolutely  pure  chemical  substance, 
the  drug  in  powder  form  will  vary  to  a  slight  extent  in  different  lots. 
Indeed,  we  doubt  whether  any  two  batches  can  be  prepared  which  are 
identical  in  all  respects.  We  have  already  stated  that  different  serial 
products  vary  in  their  oxidizability.  From  numerous  experiments 
which  we  have  carried  out  we  are  convinced  that  they  also  vary  to  a  cer- 
tain extent  in  therapeutic  properties  and  to  a  greater  degree  in  toxicity. 
Unfortunately,  these  variations  cannot  at  the  present  time  be  determined 
by  ultimate  chemical  analysis,  but  only  by  the  biologic  effects  of  the 
drug. 

With  these  prefatory  remarks  in  mind,  let  us  discuss  the  relation  of 
variations  in  the  drug  to  reactive  phenomena  following  its  use.  Ehrlich 
and  his  associates  have  repeatedly  called  attention  to  the  liability  of 
dioxydiamino-arsenobenzol  on  exposure  to  air  to  undergo  oxidation 


ADMINISTRATION    OF    SALVARSAN   AND    NEOSALVARSAN  873 

with  the  production  of  amino-oxyphenyl-arsenoxid.  This  arsenoxid 
might  be  present  in  the  drug  through  insufficient  reduction,  although 
with  care  this  is  not  apt  to  take  place. 

The  commercial  product  always  contains  some  arsenoxid,  but  it  is 
usually  less  than  1  per  cent.  Ehrlich  states  that  the  best  preparations 
will  contain  from  0.5  to  0.8  per  cent,  of  arsenoxid. 

Ehrlich  and  Bertheim1  state  that  the  toxicity  of  arsenoxid  is  very 
much  greater  than  that  of  salvarsan.  Like  other  arseno  compounds, 
the  hydrochlorid  of  dioxydiamino-arsenobenzol  possesses  the  property 
of  readily  undergoing  oxidation.  Exposed  to  the  air  it  will  soon  contain 
amino-oxyphenyl-arsenoxid;  indeed,  the  production  of  arsenoxid  com- 
pounds takes  place  if  the  preparation  is  kept  in  an  ordinary  glass 
container.  This  fact  is,  therefore,  of  the  greatest  importance  in  the 
practical  use  of  this  remedy,  because  the  amino-oxyphenyl-arsenoxid  is 
about  twenty  times  more  poisonous  than  the  pure  hydrochlorid  of  the  arseno 
compound. 

Variations  in  the  Medicament  and  their  Relation  to  the  Immediate 
Reactive  Symptoms. — We  are  firmly  convinced  that  the  complex  of 
symptoms  classed  under  group  a,  arid  characterized  by  flushing,  edema, 
etc.,  and  followed  by  pallor,  and  in  rare  instances  by  syncope,  are  due 
to  the  drug  administered  and  not  to  extraneous  causes.  Various  descrip- 
tive adjectives  have  been  applied  to  this  syndrome,  such  as  vasomotor, 
angioneurotic,  anaphylactoid,  and  nitritoid.  The  milder  grades  of  re- 
action bear  a  resemblance  to  the  symptoms  following  the  use  of  nitrate 
of  amyl,  and  the  severer  types  simulate  rather  closely  the  picture  of 
anaphylaxis.  Indeed,  Swift  maintains  that  they  represent  true  anaphy- 
lactic  phenomena.  Swift2  demonstrated  that  guinea-pigs  which  have 
been  sensitized  by  the  injection  of  a  mixture  of  guinea-pig  serum  and 
salvarsan,  and  have  been  reinjected  after  a  suitable  time  with  the  same 
mixture,  show  symptoms  like  those  seen  in  anaphylactic  shock.  Swift 
believes  that  this  phenomenon  depends  on  an  alteration  of  the  native 
serum  by  salvarsan  so  that  the  homologous  serum  acts  like  a  foreign 
protein.  It  is  pointed  out  that  the  symptoms  resembling  those  of  ana- 
phylaxis which  at  times  follow  the  administration  of  salvarsan,  occur 
usually  not  after  the  first  but  only  after  one  or  more  injections. 

The  vasomotor  symptoms  above  mentioned  we  believe  to  be  due 
to  something  in  the  drug  which  directly  or  indirectly  (perhaps  through 
anaphylaxis)  induces  a  paresis  of  the  blood-vessels,  characterized  by  dila- 
tation and  not  infrequently  by  leakage  of  the  serum  into  the  tissues.  We 

1  Berichte  d.  Deutch.  Chem.  gesell.,  vol.  45,  1,  1912,  p.  764. 

2  Jour.  Amer.  Med.  Assoc.,  1912,  lix,  1236. 


874  CHEMOTHERAPY 

regard  these  symptoms,  therefore,  as  vasoparetic  in  character.  We  do 
not  believe  that  these  symptoms  are  due  to  the  pure  molecule  of 
the  hydrochlorid  of  dioxydiamino-arsenobenzol.  From  various  experi- 
ments which  we  have  carried  out  we  strongly  suspect  that  they  are 
produced  by  traces  of  an  impurity  in  the  drug.  We  have  devoted  much 
study  to  discover  the  particular  substance  which  gives  rise  to  these 
symptoms,  but  thus  far  our  efforts  have  not  been  attended  with  success. 
We  shall  therefore  in  our  discussion  refer  to  this  substance  as  substance  X. 

Different  lots  or  batches  of  dioxydiamino-arsenobenzol  (salvarsan 
or  its  congeners)  vary  in  respect  to  the  frequency  with  which  they  induce 
the  immediate  vasomotor  or  vasoparetic  reactions.  Experienced  clin- 
icians, we  are  sure,  will  agree  that  few  or  no  reactions  of  this  character 
occur  after  the  use  of  certain  lots  of  the  drug,  and,  on  the  other  hand, 
other  batches  seem  to  be  followed  by  an  unusual  incidence  of  such 
reactions.  To  be  sure,  not  all  patients  will  exhibit  vasoparetic  reactions 
after  the  use  of  a  poor  product,  nor  will  all  remain  free  of  these  reactions 
after  the  employment  of  a  relatively  pure  product.  There  are,  doubtless, 
variations  in  susceptibility  to  substance  X,  and  some  patients  will  react 
against  the  minutest  quantity,  while  others  will  do  so  only  in  the  presence 
of  a  large  amount.  It  is  a  common  experience  for  patients  to  repeatedly 
exhibit  vasoparetic  symptoms  after  salvarsan  and  yet  remain  free  of  such 
phenomena  after  the  use  of  neosalvarsan ;  the  formaldehyd-sulphoxy- 
late  group  in  this  compound  is  attached  to  the  amino  radical,  and  the 
process  which  is  employed  seems  to  lessen  the  formation  of  substance  X. 

The  Jarisch-Herxheimer  Reaction. — This  reaction  manifests  itself 
in  the  development  of  a  rash,  the  extension  or  aggravation  of  an  existing 
eruption,  or  an  inflammatory  reaction  in  any  syphilitic  tissue  the  result 
of  treatment.  It  has  been  observed  in  the  course  of  mercurial  treatment, 
and  before  the  discovery-  of  the  Spirocheta  pallida  and  the  Wassermann 
reaction  it  was  regarded  as  of  considerable  diagnostic  importance.  Any 
aggravation  of  syphilitic  symptoms  following  the  administration  of  sal- 
varsan or  mercury  has  been  interpreted  as  a  Herxheimer  reaction.  The 
cutaneous  reaction  is  manifested  by  edema,  redness,  pain,  and  the 
mucous  patches  show  a  similar  reaction.  Gummas  become  swollen,  may 
ulcerate,  and  show  increased  exudation.  The  lancinating  pains  of  loco- 
motor  ataxia  may  be  augmented,  and  various  paralyses,  due  to  pressure, 
may  follow  in  those  nerves  that  traverse  bony  canals.  These  effects 
are  also  known  under  the  name  of  neurorelapses.  They  usually  appear 
two  or  three  months  or  even  four  or  five  months  after  treatment,  and 
they  were  at  first  believed  to  be  due  to  the  contained  arsenic  and  were 
regarded  as  constituting  a  special  danger  attending  the  use  of  salvarsan. 


ADMINISTRATION    OF    SALVARSAN    AND    NEOSALVARSAN  875 

Various  explanations  for  this  phenomenon  have  been  offered:  Ehrlich 
believes  that  it  indicates  failure  of  the  injected  dose  to  produce  complete 
destruction  of  the  spirochetes,  with  temporary  stimulation  of  the  micro- 
organisms to  increased  multiplication  and  activity.  He  very  perti- 
nently compares  the  neurorelapse  or  Herxheimer  reaction  to  the  extensive 
development  of  individual  bacterial  colonies  on  agar  plates,  when  but 
few  microorganisms  are  present,  as  contrasted  with  their  small  size 
when  the  number  is  large.  The  so-called  provocative  positive  Wasser- 
mann  reaction  may  be  considered  as  a  part  of  this  reaction. 

INTRAMUSCULAR  INJECTION 

As  was  previously  stated,  this  route  of  administration  is  not  gener- 
ally employed  at  the  present  time  because  of  the  local  irritative  effects 
of  salvarsan  especially.  When  slow  absorption  and  elimination  are  de- 
sired, or  when  intravenous  injections  are  impossible,  as  in  the  case  of 
very  young  children,  this  method  may  be  adopted.  When  salvarsan  is 
to  be  given,  the  clear,  concentrated  alkaline  solution  is  generally  used. 
Neosalvarsan  is  to  be  preferred,  because  it  is  less  irritating  than  sal- 
varsan. It  may  be  given  in  a  concentrated  aqueous  solution  or  sus- 
pended in  sterile  oil  with  the  addition  of  a  local  anesthetic,  such  as 
eucain  or  creosote,  after  this  formula:  Salvarsan,  0.4  or  0.6  gm.;  creosote 
(beechwood),  gtt.  ij ;  sterile  liquid  petrolatum,  5  c.c.  Mix  well  in  a  sterile 
mortar.  The  injections  are  made,  under  strict  antiseptic  precautions, 
in  the  gluteal  region,  into  the  upper  and  outer  quadrant  of  the  muscles. 
The  syringe  and  the  needle  should  be  sterilized  and  the  injection  pre- 
pared. After  cleansing  the  skin  with  alcohol  the  needle  is  quickly 
and  boldly  plunged  into  the  deep  tissues.  The  syringe  may  then  be 
detached  to  ascertain  if  blood  flows,  which  would  show  that  a  vein  has 
been  punctured  and  require  a  reinsertion;  if  blood  does  not  appear, 
the  barrel  should  be  reattached  and  the  injection  slowly  given. 

Intraspinous  Injection  of  Neosalvarsan. — Owing  to  the  fact  that  a 
drug  injected  intravenously  may  not  reach  the  tissues  of  the  central 
nervous  system,  salvarsan  and  neosalvarsan  have  not  fulfilled  the  early 
expectations,  apparently  chiefly  for  the  reason  that  they  cannot  reach 
the  spirochetes.  It  would  seem,  therefore,  that  adequate  treatment  of 
syphilis  of  the  central  nervous  system  consists  in  the  direct  application 
of  the  remedy  to  the  infected  tissues  themselves.  Wechselmann  l  and 
Marinesco  2  have  injected  salvarsan,  and  Marie  and  Levaditi 3  and  others 

1  Deutsch.  med.  Wochenschr.,  1912,  38,  1446. 

2  Zeitschr.  f.  phys.  u.  Therap.,  1913,  17,  194. 

3  Bull,  et  Soc.  med.  d.  h6p.,  Paris,  November  18,  1913. 


876  CHEMOTHERAPY 

have  given  neosalvarsan,  directly  into  the  spinal  canal.  The  results, 
however,  were  either  dubious  or  frankly  dangerous,  and  the  injections 
were  followed  by  severe  and  alarming  symptoms,  due  mainly  to  the  pro- 
duction by  the  drug  of  direct  irritation  upon  the  sensitive  nervous  sys- 
tem. Ravaut1  has  found  that  the  use  of  hypertonic  solutions  of  neo- 
salvarsan are  better  borne  and  of  value  in  the  treatment  of  special  cases. 
Wile 2  has  also  employed  this  method,  and  has  found  it  of  some  value  in 
cerebrospinal  syphilis.  Tabetics  presenting  no  bladder  or  rectal  symp- 
toms were  found  to  do  especially  well.  As  is  to  be  expected,  the  earlier 
the  treatment  is  instituted,  the  better  are  the  results.  This  form  of 
treatment  is,  however,  to  be  regarded  as  dangerous,  and  is  to  be  used 
only  in  selected  cases,  and  with  a  full  understanding  of  the  risks  incurred. 
Wile  has  given  the  following  technic : 

"The  solution  used  for  injection  consists  of  a  6  per  cent,  solution  of  neosalvarsan 
in  distilled  water.  This  solution  is  hypertonic,  and  made  of  such  concentration  that 
each  minim  must  contain  3  mg.  of  the  drug.  The  dosage  injected  is  from  3  to  12 
mg. — that  is,  from  one  to  four  drops  of  the  solution,  which  is  made  up  as  follows: 

"An  ampule  containing  0.3  mg.  of  neosalvarsan  is  dissolved  in  5  c.c.  of  freshly 
distilled  water.  If  the  ampule  contains  0.6  gm.,  10  c.c.  of  water  are  used.  In  both 
solutions  each  drop  will  contain  3  mg.  of  the  drug.  The  syringe  employed  for  the 
injection  is  accurately  graduated  in  drops.  The  patient  is  placed  in  a  position  for 
lumbar  puncture,  either  sitting  or  lying,  according  to  the  choice  of  the  operator. 
The  puncture  is  then  made  with  the  needle,  the  end  of  which  fits  the  graduated 
syringe.  After  a  few  drops  of  the  spinal  fluid  have  flowed  out  of  the  cannula,  or  a 
greater  quantity  if  a  diagnostic  puncture  is  desired  at  this  time,  the  syringe  is  fitted 
into  the  needle,  and  the  fluid  is  allowed  to  run  back  into  the  syringe  barrel,  thus 
mixing  with  the  amount  of  the  drug  in  the  barrel.  The  mixed  spinal  fluid  and  drug 
are  then  gently  forced  into  the  canal,  and  slight  suction  is  made  on  the  syringe  to 
withdraw  a  second  amount  of  fluid,  which  washes  out  the  needle.  This  is  then  reintro- 
duced,  the  needle  is  quickly  withdrawn,  and  the  patient  placed  in  the  Trendelenburg 
position,  in  which  position  he  is  allowed  to  remain  for  at  least  one  hour." 

INTRASPINOUS  INJECTION  OF  SALVARSANIZED  SERUM 

In  this  method  the  salvarsan  or  neosalvarsan  is  injected  intrave- 
nously in  the  usual  manner,  and  shortly  afterward  blood  is  withdrawn, 
the  serum  separated,  heated  to  55°  C.  for  half  an  hour,  and  a  portion 
injected  intraspinously.  In  this  technic,  which  has  been  worked  out 
largely  by  Swift  and  Ellis,  the  drug  is  highly  diluted,  and  therefore  is 
not  likely  to  produce  much  irritation.  In  addition,  some  of  the  good 
effects  may  be  due  to  the  presence  of  antibodies  in  the  serum  itself. 
This  method  constitutes  the  most  useful  form  of  intraspinous  medica- 

1  Ann.  de  Med.,  1914,  1,  49. 

2  Jour.  Amer.  Med.  Assoc.,  1914,  Ixii,  1165;  ibid.,  1914,  Ixiii,  137. 


ADMINISTRATION   OF   SALVARSAN   AND    NEOSALVARSAN       877 

tion  thus  far  proposed.     The  details  of  this  technic  are  given  in  Chapter 
XXXI. 

Mention  has  also  been  made,  in  the  preceding  chapter,  of  a  method 
of  intraspinous  medication  consisting  in  the  injection  of  a  combination 
of  the  patient's  serum  and  salvarsan  or  neosalvarsan  mixed  in  vitro. 
This  method  is  to  be  regarded  as  more  dangerous  and  probably  less 
efficient  than  that  of  Swift  and  Ellis,  and  as  yet  in  the  experimental 
stage.  Recently  Fordyce  *  has  given  the  following  technic  (Ogilvie) : 

"Fifty  c.c.  of  blood  are  drawn  into  a  centrifuge  bottle  and  centrifuged  twice. 
It  is  important  to  have  the  serum  clear  and  free  from  fibrin  and  blood-cells.  To 
obtain  the  requisite  amount  of  the  drug  old  salvarsan  is  mixed  in  the  usual  way,  in 
the  proportion  of  0.1  gm.  to  40  c.c.  of  fluid,  care  being  taken  not  to  overalkalinize; 
0.4  c.c.  of  this  solution  is  the  equivalent  of  1  mg.,  and  is  taken  as  the  standard  for 
measuring  the  dosage.  For  this  purpose  a  1  c.c.  pipet  graduated  in  hundredths  should 
be  employed.  The  desired  amount  of  salvarsan  is  added  to  from  12  to  15  c.c.  of  the 
serum,  shaken  to  and  fro  to  mix  thoroughly,  and  then  placed  in  the  incubator  at 
37°  C.  (98.6°  F.)  for  one  hour,  after  which  it  is  inactivated  for  half  an  hour  at  56°  C. 
(132.8°  F.).  The  latter  is  the  most  important  step  in  the  technic;  the  spirocheticidal 
properties  of  the  serum  are  markedly  increased  by  heating. 

"Salvarsanized  serum,  prepared  according  to  this  method,  must  be  used  fresh, 
that  is,  within  three  hours  of  the  time  that  it  is  made  up.  Patients  should  be  prepared 
for  its  administration  as  for  intravenous  injection,  with  a  laxative  the  night  before 
and  only  a  light  meal  two  hours  prior  to  the  treatment. 

"A  lumbar  puncture  is  made,  and  an  amount  of  fluid  equivalent  to  the  amount 
introduced  is  withdrawn.  While  the  needle  is  in  situ,  the  barrel  of  a  Luer  syringe  is 
connected  by  means  of  a  piece  of  rubber  tubing.  Spinal  fluid  is  allowed  to  fill  this 
to  expel  the  air,  and  the  serum  is  then  permitted  to  flow  in  by  gravity.  After  its 
administration  the  patient  should  lie  flat  without  any  pillows,  the  foot  of  the  bed 
being  kept  elevated  for  several  hours.  He  must  be  kept  in  bed  for  at  least  twenty- 
four  hours,  in  some  cases  forty-eight  or  seventy-two  hours.  Failure  to  do  this  may 
result  in  unpleasant  symptoms,  as  pain  in  the  extremities,  headache,  anesthesia, 
and  bladder  and  rectal  paresis." 

The  proper  dose  is  still  a  matter  of  doubt,  and  a  note  of  warning  must 
be  sounded  against  employing  too  large  amounts.  Fordyce  believes 
that  the  limit  of  safety  lies  within  0.5  mg.  It  is  better  to  begin  with  a 
dose  of  0.25  mg.,  repeating  or  gradually  increasing  this  according  to  the 
tolerance  of  the  patient.  The  intervals  between  doses  should  be  two 
weeks  or  longer. 

In  the  treatment  of  syphilis  of  the  central  nervous  system  the  in- 
travenous method  may  first  be  tried,  giving  a  series  of  six  or  eight  in- 
jections of  salvarsan,  beginning  with  a  dose  of  from  0.25  to  0.3  gm.,  and 
injecting  it  at  intervals  of  from  one  to  two  weeks.  At  the  end  of  this 
time  the  blood  and  spinal  fluid  should  be  tested,  and  if  it  is  found  that 
1  Jour.  Amer.  Med.  Assoc.,  1914,  Ixiii,  552. 


878  CHEMOTHERAPY 

no  marked  effect  has  been  obtained  either  clinically  or  serologically, 
a  course  of  intraspinous  injections  may  be  considered. 

CONTRAINDICATIONS  AND  PRECAUTIONS  IN  SALVARSAN  THERAPY 
When  it  is  remembered  that  millions  of  doses  of  salvarsan  and  neo- 
salvarsan  have  been  given  by  thousands  of  different  physicians,  by 
many  and  diverse  methods,  the  relative  safety  of  the  drug  is  apparent. 
The  fact  remains,  nevertheless,  that  patients  have  succumbed  soon 
after  receiving  an  injection  who  would  not  otherwise  have  died  at  that 
time.  These  fatalities  cannot  be  explained  on  a  purely  toxicologic 
basis.  Recently  Wechselmann1  has  reviewed  the  deaths  following 
salvarsan  therapy  and  has  arrived  at  the  conclusion  that  "insufficiency 
of  the  kidney,  and  not  hyper  sensitiveness  of  the  brain,  is  the  point  of  the 
entire  question  of  salvarsan  fatalities. "  He  is  very  emphatic  in  warning 
against  mixed  mercurial  and  salvarsan  treatment,  especially  if  salvarsan 
is  given  after  a  course  of  mercury,  for  the  latter  drug  is  a  renal  irritant 
and  may  lead  to  prolonged  retention  of  the  salvarsan,  which  may  undergo 
a  process  of  reduction  in  the  tissues  and  form  one  of  the  poisonous 
products  that  Ehrlich  has  called  "arsenoxid." 

Contraindications  to  salvarsan  therapy  may  be  divided  into  two 
main  groups: 

1.  Those  to  whom  the  injection  may  be  dangerous  on  account  of 
the  reaction  that  may  follow,  as,  for  example,  cases  of  early  cerebral 
lues  with  cranial  nerve  manifestations  of  an  exudative  character;    also 
cases  of  tabes  with  beginning  optic  atrophy.     In  these  patients  salvar- 
san or  neosalvarsan  should  be  given  with  extreme  caution  and  only  in 
small  doses,  and  frequent  examinations  of  the  eyes  should  be  made. 

2.  Those  with  extensive  disease  of  the  circulatory  system,  such  as 
severe  uncompensated  heart  disease,  coronary  sclerosis,  and  extensive 
aneurysm;    also  cases  of  diabetes  mellitus,  severe  nephritis,  ulceration 
of  the  stomach,  and  advanced  tuberculosis  or  carcinoma. 

In  this  connection  I  may  cite  here  the  precautions  laid  down  by 
Wechselmann,  director  of  the  dermatological  department  of  the  Rudolf 
Virchow  Hospital,  in  whose  clinic  over  25,000  doses  of  salvarsan  have 
been  given.  These  precautions  are: 

1.  To  use  the  most  exact  technic. 

2.  To  employ  a  dose  of  the  drug  carefully  adapted  to  the  individual 

case. 

luThe  Pathogenesis  of  Salvarsan  Fatalities,"  translated  by  Martin,  1913, 
Fleming  Smith  Co.,  St.  Louis. 


VALUE    OF   SALVARSAN   AND    NEOSALVARSAN    IN    SYPHILIS     879 

3.  To  make  careful  observation  by  the  most  exact  chemical  and 

microscopic  examination  of  the  urinary  secretion  when  em- 
ploying salvarsan.  This  holds  good  particularly  when  the  com- 
bined treatment  is  employed. 

4.  The  conjoint  use  of  salvarsan  with  heavy  mercurial  treatment  is 

dangerous!  If  one  will  use  the  combined  treatment,  then  let 
him  give  mercury  very  carefully  many  days  after  giving  the 
last  salvarsan  injection,  but  he  should  not  reverse  this  rule. 

5.  Consider  carefully  every  general  reaction  or  rise  of  temperature 

following  the  use  of  salvarsan,  and  make  a  thorough  investiga- 
tion as  to  its  causes. 


VALUE  OF  SALVARSAN  AND  NEOSALVARSAN  IN  THE  TREATMENT  OF 

SYPHILIS 

It  is  too  soon  to  judge  of  the  ultimate  value  of  salvarsan  in  the  treat- 
ment of  syphilis.  It  may  be  stated,  however,  that  the  immediate  effects 
are  so  beneficial  that  the  drug  is  to  be  regarded  as  the  best  we  possess 
for  the  treatment  of  lues.  As  is  to  be  expected,  the  best  results  are  se- 
cured when  the  remedy  is  administered  early,  and,  of  course,  when  tissue 
changes  have  occurred  no  drug  can  be  expected  to  restore  a  lost  func- 
tion, although  it  may  bring  about  considerable  improvement  by  causing 
the  disappearance  of  syphilitic  tissue.  In  long-standing  cases  salvarsan 
may,  at  least,  hold  the  symptoms  in  abeyance,  and  effectively  prevent 
progress  of  the  disease.  It  is  especially  valuable  in  patients  who  cannot 
tolerate  mercury.  The  remarkable  efficacy  of  salvarsan  is  attested  by 
the  rapidity  with  which  spirochetes  disappear  from  primary  sores  and 
from  secondary  lesions,  the  manner  in  which  they  disappear  being  fre- 
quently little  short  of  marvelous.  There  can  be  no  doubt  but  that  sal- 
varsan is  a  powerful  spirocheticide,  having  but  remarkably  little  toxic 
effect  upon  the  body-cells.  It  would  appear  that  in  order  to  rid  the 
tissues  of  the  spirochetes  it  is  only  necessary  to  bring  the  drug  into  con- 
tact with  them.  For  this  reason  newer  and  better  methods  of  adminis- 
tration are  bound  to  increase  the  value  of  the  drug.  Barring  the  well- 
recognized  contraindications,  salvarsan  may  be  used  in  practically  all 
stages  of  the  disease.  In  the  later  stages  of  the  infection,  in  which  the 
central  nervous  tissue  is  involved,  the  physician  must  bear  in  mind  the 
possible  danger  of  a  neurorelapse  or  a  Herxheimer  reaction  occurring. 
While  salvarsan  may  do  no  good  in  late  cases,  since  it  cannot  restore  lost 
nerve  tissue,  it  may  alleviate  symptoms  and  prevent  progress  of  the  in- 


880  CHEMOTHERAPY 

fection.  In  the  treatment  of  any  case  of  syphilis  the  best  results  are  not 
secured  from  following  any  course  of  treatment  according  to  a  rule  of 
thumb;  they  are  obtained  only  by  a  suitable  combination  of  expert 
clinical  knowledge  and  training  in  serologic  technic.  All  possible  aid 
should  be  enlisted  in  the  treatment  of  the  disease. 

It  is  beyond  the  scope  of  this  book  to  present  either  case  reports  or  a 
lengthy  discussion  of  salvarsan  in  the  treatment  of  the  various  stages  of 
syphilis;  suffice  it  to  say  that,  in  the  absence  of  contraindications,  the 
drug  may  be  administered  whenever  living  spirochetes  are  present  in  a 
patient's  tissues,  as  evidenced  either  by  clinical  symptoms  or  a  positive 
Wassermann  reaction.  The  method  of  administration  selected  should 
be  that  which  will  best  bring  the  drug  into  contact  with  the  diseased 
tissues. 

In  the  studies  of  Schamberg,  Raiziss,  and  I1  upon  the  chemotherapy 
of  mercurial  compounds  as  compared  with  salvarsan,  salvarsan  has 
shown  itself  to  be  by  far  the  most  powerful  trypanocide  known  in  both 
the  living  animal  and  in  the  test-tube.  In  my  in  vitro-vivo  method2  it 
destroys  trypanosomes  in  dilution  as  high  as  1  : 40,000.  Bichlorid  of 
mercury  shows  markedly  inferior  values  in  this  respect.  The  superiority 
of  the  influence  of  salvarsan  over  mercury  in  experimental  trypanoso- 
miasis  is  incontestable.  In  the  test-tube  salvarsan  exhibits  a  greater 
destructive  influence  on  animal  parasites,  and  mercury  a  greater  de- 
structive influence  on  vegetable  parasites.  Salvarsan  is  a  powerful 
trypanocide  and  a  feeble  bactericide;  mercury  is  a  powerful  bactericide 
and  a  relatively  feeble  trypanocide. 


SALVARSAN  IN  THE  TREATMENT  OF  NON-SYPHILITIC  DISEASES 
While  salvarsan  therapy  has  achieved  its  greatest  success  in  the 
treatment  of  syphilis,  its  influence  on  certain  non-syphilitic  diseases  has 
been  so  pronounced  that  it  is  being  used  in  an  ever-increasing  number  of 
diseases,  the  aim  being  to  thus  enlarge  its  sphere  of  usefulness.  A  re- 
cent systematic  study  of  the  subject  has  been  made  by  Best.3  Good  re- 
sults have  been  reported  by  various  observers  in  the  following  diseases : 
Frambesia  (Yaws). — The  results  achieved  in  this  disease  from  the 
use  of  salvarsan  have  equaled  those  obtained  in  syphilis.  Two  thousand 
four  hundred  and  thirty  cases  have  been  reported,  a  cure  having  been 
effected  in  all  but  a  few  instances.  Since  the  use  of  salvarsan  has  come 
into  favor  hospitals  for  the  treatment  of  frambesia  have  been  closed. 

1  Amer.  Jour.  Syphilis,  1917,  1,  3.  2  Jour.  Infect.  Dis.,  1917,  20,  10. 

3  Jour.  Amer.  Med.  Assoc.,  1914,  bdii,  375. 


CHEMOTHERAPY    IN    BACTERIAL   DISEASES  881 

Relapsing  Fever. — In  the  treatment  of  this  disease  the  drug  has 
likewise  had  a  remarkable  effect.  Of  195  cases  reported,  in  most  of  them 
the  microorganisms  could  not  be  found  in  the  blood  after  an  injection, 
and  in  none  of  the  cases  were  relapses  observed  to  occur. 

Filariasis. — Salvarsan  has  been  found  effective  in  killing  the  Filaria 
sanguinis  hominis  and  in  ridding  the  blood  of  these  parasites.  Of 
course,  in  elephantiasis  it  is  impossible  to  restore  the  affected  parts  to 
their  normal  appearance. 

Vincent's  Angina. — An  immediate  improvement  and  rapid  healing 
have  been  reported  as  following  either  an  intravenous  injection  or  the 
local  application  of  the  drug  in  the  form  of  a  dusting-powder  or  in  sus- 
pension in  glycerin. 

Duhring's  Disease  (Dermatitis  Herpetiformis),  Scurvy,  Chorea,  Ma- 
laria, Acanthosis  Nigricans,  Ulcus  Tropicum,  Variola,  and  Verruca  Plana. 
— In  all  these  affections  salvarsan  has  been  found  to  exert  a  beneficial 
and  curative  effect,  either  by  direct  action  upon  the  microorganisms 
present  or  as  the  result  of  the  alterative  and  stimulating  effect  of  the 
arsenic. 

In  Aleppo  boil,  leprosy,  lupus  vulgaris,  tuberculosis,  anemia,  kera- 
tosis  follicularis,  lichen  planus,  mycosis  fungoides,  pellagra,  and  pity- 
riasis  rubra,  good  or  indifferent  results  have  been  reported.  In 
many  conditions  it  would  appear  that  salvarsan  exerts  a  direct  germi- 
cidal  effect,  and  in  others  the  beneficial  results  appear  to  be  dependent 
upon  a  certain  tonic,  stimulating,  and  alterative  effect  of  the  arsenic. 
In  chancroid,  scarlet  fever,  Hodgkin's  disease,  psoriasis,  trichinosis,  and 
trypanosomiasis  the  drug  does  not  appear  to  exercise  any  influence, 
which  is  due  probably,  in  the  last-mentioned  disease,  to  the  fact  that 
trypanosomes  are  much  more  likely  to  become  arsenic-fast  than  are  the 
spirochetes. 

CHEMOTHERAPY  IN  BACTERIAL  DISEASES 

Principles. — The  ultimate  aim  in  bactericidal  chemotherapy  is  the 
discovery  or  the  rational  and  systematic  development  by  synthesis  of 
a  substance  that  is  strictly  monotropic ;  that  is,  one  possessing  a  selective 
affinity  for  the  protoplasm  of  a  particular  microparasite  and  exerting 
a  specific  killing  effect  on  that  organism.  Thus  far  this  desideratum  has 
not  been  achieved ;  as  even  arsenobenzol  (salvarsan)  is  not  strictly  para- 
sitotropic  for  Treponema  pallidum,  but  possesses  also  a  marked  para- 
sitropism  for  other  spirochetes  and  other  protozoa,  notably  various 

trypanosomes.    While  the  development  or  discovery  of  substances  which 
56 


882  CHEMOTHERAPY 

are  markedly  bacteriotropic  or  protozootropic  in  general  constitutes  an 
important  advance,  the  ultimate  aim,  as  stated,  should  be  the  synthesis 
of  a  strictly  monotropic  substance.  It  is  suggested  that  further  studies 
with  arsenobenzol,  tending  to  increase  its  parasitotropic  effect  on  spiro- 
chetes  alone  and  Treponema  pallidum  in  particular,  will  still  further 
increase  the  therapeutic  efficacy  of  this  drug. 

"Leads"  in  Chemotherapeutic  Research. — In  the  present  state  of  our 
knowledge  of  chemotherapy  chance  or  accidental  discovery  must  play 
an  important  role  in  the  discovery  of  a  "lead."  Substances  are  to  be 
selected  or  prepared  on  as  systematic  a  basis  as  possible,  and  tried  out  by 
actual  experiment;  those  yielding  encouraging  results  are  then  subjected 
to  various  systematic  modifications  with  experimental  trial  of  the  new 
compounds.  In  this  manner  chemotherapeutic  research  proves  to  be 
costly  and  laborious,  as  amply  demonstrated  by  the  prolonged  and 
costly  series  of  experiments  directed  by  Ehrlich,  which  resulted  in  the 
discovery  of  arsenobenzol. 

In  the  discovery  of  leads  and  the  study  of  new  compounds  animal 
experiments  are  of  primary  importance,  not  only  because  these  are  the 
sole  means  of  determining  the  organotropic  or  toxic  effects  of  the  com- 
pounds, but  because  they  are  the  sole  means  of  determining  the  actual 
parasitotropic  or  therapeutic  effects.  In  conducting  these  experiments 
it  is  necessary  to  select  a  protozoon  or  bacterium  that  yields  a  uniform 
infection  of  the  animals  of  not  too  severe  a  character,  and  to  reproduce 
as  far  as  possible  the  same  lesions  in  the  animals  as  are  found  in  man. 
The  test  microparasite  should  either  produce  definite  lesions  easy  of 
detection  and  study  or  cause  the  death  of  the  animal  in  a  given  period 
of  time.  For  studies  in  bacterial  chemotherapy  virulent  cultures  of 
the  pneumococcus  are  admirably  adapted  to  work  with  mice  and  rabbits; 
in  studies  of  protozoa  Trypanosoma  equiperdum  or  T.  brucei  is  valu- 
able, the  white  rat  being  used  as  host. 

These  experiments,  however,  are  likely  to  prove  costly,  and,  as  in  the 
«ase  of  tuberculosis,  it  may  be  many  weeks  or  months  before  results  can 
be  determined.  Furthermore,  special  animals,  such  as  monkeys  or  the 
higher  apes,  may  be  necessary  for  the  determination  of  the  parasitotropic 
effect  of  a  substance,  as  in  the  case  of  anterior  poliomyelitis  and  syphilis, 
in  which  the  particular  microparasites  fail  entirely  to  infect  such  animals 
as  rabbits,  guinea-pigs,  and  rats,  or,  at  least,  fail  to  do  so  with  sufficient 
uniformity.  In  chemotherapeutic  studies  in  syphilis  the  rabbit  may  be 
employed,  although  the  infection  usually  pursues  in  this  animal  a  brief 
course  tending  to  spontaneous  recovery.  For  these  reasons  an  effort 


CHEMOTHERAPY    IN    BACTERIAL   DISEASES  883 

should  be  made  to  train  or  adapt  a  race  of  the  particular  microparasite 
under  study  to  survive  and  multiply  in  the  tissues  of  an  animal  easily 
obtained  and  handled,  so  that  the  abundance  of  experimental  material 
needed  in  chemotherapeutic  studies  on  a  large  scale  may  be  available. 
In  addition  to  this,  as  stated,  an  effort  should  be  made  as  far  as  possible 
to  reproduce  in  the  experimental  animals  lesions  similar  to  those  found 
in  man.  For  example,  the  effect  of  a  drug  in  the  blood  of  a  rabbit  or 
white  mouse  in  a  pneumococcus  bacteremia  must  be  different  from  that 
in  the  exudate  of  a  consolidated  lung  in  pneumonia  of  man. 

The  Role  of  Bactericidal  Tests  in  the  Living  Animal  in  Bacterial 
Chemotherapy. — In  chemotherapeutic  studies  on  syphilis  experience  has 
shown  that  a  trypanosome  may  be  used,  as  Trypanosoma  equiperdum, 
or  a  spirochete  other  than  Spirochseta  pallida,  as  S.  gallinarum,  to  deter- 
mine the  parasitotropic  effect  of  new  compounds  as  they  are  produced; 
because  those  compounds  showing  marked  effects  on  these  micropara- 
sites,  as,  for  example,  arsenobenzol,  also  produce  a  profound  effect  on  S. 
pallida  in  human  and  experimental  syphilis.  It  remains  to  be  determined, 
however,  whether  similar  conditions  hold  true  in  the  chemotherapy  of 
bacterial  infections;  that  is,  whether  a  compound  showing  a  high  bacteri- 
cidal effect  in  vitro,  or  even  in  vivo,  on  one  microorganism,  as  Bacillus 
typhosus  or  the  pneumococcus,  will  show  a  similar  effect  on  the  micro- 
organisms of  other  diseases,  as  tuberculosis  or  anterior  poliomyelitis. 
Suffice  it  to  say  that  the  ultimate  value  of  any  drug  can  be  determined 
only  by  tests  in  the  lower  animals;  first  to  determine  toxicity,  and  then 
therapeutic  value.  If  these  tests  show  a  low  toxicity  and  a  promising 
therapeutic  effect  the  court  of  last  resort  is  the  use  of  the  compound  in 
the  treatment  of  man. 

The  Role  of  Bactericidal  Tests  in  Vitro  in  Bacterial  Chemotherapy — 
In  chemotherapeutic  studies  on  bacterial  infections  experiments  in  vitro- 
may  be  said  to  have  a  positive  value  in  preliminary  orientation  in  the 
development  of  leads  and  the  study  of  new  compounds  as  they  are  pro- 
duced. Experimental  data  at  hand  tend  to  show  that  substances  pos- 
sessing a  high  bactericidal  activity  in  vitro,  and  particularly  in  a  men- 
struum of  fresh  sterile  serum,  are  more  likely  to  exert  an  inhibitory  effect  in- 
vivo;  for  example,  Morgenroth's  drug,  optochin  (ethylhydrocuprein)  and 
its  hydrochlorid,  exert  a  very  high  bactericidal  action  on  the  pneumococcus 
in  vitro,  and  are  likewise  effective  to  some  extent  in  vivo;  other  cinchona, 
derivatives,  including  certain  salts  of  quinin,  possessing  more  or  less 
bactericidal  value  in  vitro  are  likewise  effective  to  a  certain  degree  in. 
vivo.1  Arsenobenzol  has  been  found  to  possess  the  highest  parasitotropic 
1  Jour.  Infect.  Dis.,  1917,  20,  81. 


884  CHEMOTHERAPY 

activity  on  Trypanosoma  equiperdum  in  vitro  of  a  number  of  substances 
tested,1  and,  as  well  known,  this  drug  exerts  the  best  therapeutic  or 
parasitotropic  effects  in  vivo.  Unfortunately,  however,  other  substances 
that  are  highly  bactericidal  in  vitro,  as  the  mercurials,  also  possess  a 
high  degree  of  toxicity  for  the  living  animal,  and  all  efforts  of  the  chemo- 
therapeutist  have  so  far  failed  to  lower  materially  the  toxicity  of  these 
compounds. 

Since  the  time  of  the  earliest  discoveries  in  bacteriology  and  of 
bactericides  substances  highly  bactericidal  in  the  test-tube  have  been 
known  to  fail  to  exert  any  appreciable  influence  in  vivo  when  given  in 
safe  doses.  This  lack  of  effect  may  be  due  to  the  high  organotropic  or 
toxic  effect  of  the  substance  on  the  body  cells  in  general  or  on  a  particular 
group  of  cells  of  vital  centers,  precluding  the  use  of  a  bactericidal  quan- 
tity; to  insolubility  and  difficulty  of  administration;  to  rapid  union  with 
the  proteins  of  the  body  and  the  formation  of  inert  compounds;  to  rapid 
elimination;  to  failure  to  reach  the  microorganism  in  a  lesion,  and  to 
still  other  causes.  Of  these  possibilities,  the  first  mentioned  is  of  primary 
importance;  but  the  method  and  manner  of  attack  of  the  bactericidal 
drugs  on  the  protoplasm  of  cells,  aside  from  coagulation,  is  almost  un- 
known. Nevertheless,  it  is  possible  by  systematic  modification  of  the 
molecule  through  addition,  removal,  or  substitution  of  certain  atom 
groups,  to  produce  in  some  instances  a  sufficient  lowering  of  toxicity 
to  give  an  available  therapeutic  agent.  The  demonstration  of  this  fact 
constitutes  the  great  triumph  of  Ehrlich  and  opens  a  fascinating  field 
of  research  to  chemist  and  biologist. 

It  is  highly  probable  that  experimental  studies  tending  to  increase 
the  monotropism  of  a  drug  in  vitro  and  particularly  in  a  menstruum  of 
serum  will  prove  of  value  in  chemotherapeutic  work.  For  example, 
ethylhydrocuprein,  which  shows  the  highest  selective  action  upon  the 
pneumococcus  in  vitro,  likewise  proves  mos  btactericidal  in  vivo.  Simi- 
lar facts  may  be  proved  in  the  future  in  connection  with  the  micropara- 
sites  of  tuberculosis  and  of  typhoid  fever,  staphylococci,  and  other 
infective  bacteria. 

On  the  other  hand,  a  substance  failing  to  exert  parasitotropic  action 
either  in  vitro  or  in  vivo  may  still  prove  of  value  as  a  basis  of  composition, 
and  offer  a  valuable  lead  in  chemotherapeutic  research.  For  example, 
arsenic  in  the  form  of  the  trioxid  has  no  appreciable  effect  on  Trypano- 
soma equiperdum  in  vivo  and  but  slight  effect  in  vitro,2  and  yet  it  forms 
the  basis  of  arsenobenzol,  which  is  so  highly  parasitotropic  both  in  vitro 
and  in  vivo. 

1  Jour.  Infect.  Dis.,  1917,  20,  10.  2  Jour.  Infect.  Dis.,  1917,  20,  10. 


CHEMOTHERAPY    IN    BACTERIAL   DISEASES  885 

Likewise,  it  is  possible  that  a  chemical  may  be  more  efficacious  in  vivo 
than  in  vitro  by  reason  of  the  formation  of  new  and  more  active  com- 
pounds in  vivo]  or  by  exciting  the  body  cells  to  produce  antibodies  for 
the  parasitic  antigen  or  chemically  facilitating  such  production;  or  by 
the  stimulation  of  phagocytosis,  as  suggested  by  some  of  our  experiments 
in  the  case  of  quinin  compounds  in  pneumococcus  infections.1  Still,  as 
a  general  rule,  it  appears  that  substances  without  appreciable  effect  in 
vitro  are  likely  to  be  similarly  inert  in  uivo.  Arsenobenzol  offers  no 
exception  to  the  rule;  contrary  to  the  general  impression,  it  possesses 
a  marked  trypanocidal  activity  in  vitro. 

It  is  highly  probable,  therefore,  that  experiments  in  vitro  have  a 
definite  value  in  chemotherapeutic  research  as  methods  of  preliminary 
orientation  and  in  the  development  of  monotropic  chemicals.  This 
value  is  greater  when  the  identical  microparasite  causing  the  definite 
infection  under  study  is  employed  in  the  tests.  In  the  absence  of  a  pure 
culture  of  the  particular  microparasite  against  which  a  destroyer  is 
sought,  or  in  the  presence  of  insuperable  technical  difficulties,  other 
and  more  easily  cultivated  organisms  closely  or  remotely  related,  or 
even  of  a  different  biologic  order,  may  be  employed,  as  in  the  use  of 
Bacillus  typhosus  and  other  bacteria  by  Jacobs  and  his  colleagues2 
in  chemotherapeutic  studies  concerning  anterior  poliomyelitis. 

Pneumococcus  Infections. — As  has  previously  been  stated  in  Chapter 
XXIX,  progress  in  the  chemotherapy  of  pneumococcus  infections 
has  been  made  by  Lamar  in  the  treatment  of  pneumococcus  infections 
with  mixtures  of  pneumococcus  immune  serum,  sodium  oleate,  and  boric 
acid.  Particularly  valuable  work  in  this  field  has  been  done  by  Morgen- 
roth  and  his  associates. 

Influenced  less  by  this  tradition  than  by  the  effects  of  the  use  of 
quinin  compounds  as  observed  in  experimental  trypanosomiasis,  and 
by  the  fact  that  certain  peculiar  biologic  relationships  (as  bile  solubility) 
have  been  found  by  Schilling3  and  Neufeld4  to  exist  between  trypano- 
somes  and  spirilla,  on  the  one  hand,  and  pneumococci  on  the  other, 
Morgenroth  and  Levy5  chose  quinin  and  its  derivatives  as  a  basis  and 
lead  for  chemotherapeutic  studies  on  experimental  pneumococcus  infec- 
tions. Best  results  were  observed  with  optochin  (ethylhydrocuprein), 
a  synthetic  derivative  of  hydroquinin  (methylhydrocuprein),  which 

1  Jour.  Infect.  Dis.,  1917,  20,  101.  2  Jour.  Exper.  Med.,  1916,  23,  569. 

3  Centralbl.  f.  BakterioL,  I,  O.,  1902,  31,  452. 

4  Arb.  a.  d.  k.  Gendhtsamte,  1907,  25,  494;  Ztschr.  f.  Hyg.  u.  Infektionskr.,  1900, 
34,  454. 

5  Berl.  klin.  Wchnschr.,  1911,  48,  1560,  1979. 


886  CHEMOTHERAPY 

latter  exists  in  the  cinchona  bark  or  can  be  prepared  synthetically.  In 
experimental  infections  of  mice  the  new  drug  (optochin)  was  found  to 
prevent  the  development  of  infection  in  some  90  per  cent,  of  animals  in 
which  treatment  was  postponed  till  after  inoculation.  Later  Guttman,1 
Morgenroth  and  Kaufman,2  and  Levy3  reported  further  experiments 
indicating  the  protective  and  curative  action  of  ethylhydrocuprein  (base) 
and  its  hydrochlorid  in  experimental  infections  in  mice.  Wright4  found 
that  the  administration  of  ethylhydrocuprein  to  man  resulted  in  an 
increased  bactericidal  power  of  the  blood.  These  observations  were 
confirmed  by  Moore5  with  animals.  Moore6  also  reported  that  ethyl- 
hydrocuprein exerts  a  well-marked  protective  action  against  experi- 
mental pneumococcal  infection  in  mice  in  the  case  of  type  strains  of 
all  four  groups  of  pneumococci;  this  protective  action  was  found  efficient 
against  many  multiples  of  the  minimal  lethal  dose.  Rosenthal7  gives 
an  excellent  review  of  the  literature  bearing  on  the  chemotherapeutic 
treatment  of  pneumonia,  leading  up  to  and  including  the  work  of  Morgen- 
roth and  Levy  and  others.  Cohen,  Heist,  and  I8  found  that  in  mice  the 
intravenous  injection  of  ethylhydrocuprein  hydrochlorid  in  amounts 
ranging  from  0.02  gm.  and  higher  per  kilo  of  body  weight  affords  com- 
plete protection  against  50  minimal  lethal  doses  of  pneumococci  given 
two  hours  later  by  intraperitoneal  injection.  Doses  ranging  from  0.01 
to  0.006  gm.  afford  partial  protection.  Among  rabbits  a  dose  of  at  least 
0.01  gm.  of  ethylhydrocuprein  per  kilo  of  body  weight  is  required  to 
afford  protection  against  fifty  minimal  lethal  doses  of  pneumococci 
given  two  hours  later  by  intravenous  injection. 

Clinically,  the  local  application  of  a  1  to  2  per  cent,  solution  of  ethyl- 
hydrocuprein hydrochlorid  has  been  reported  by  a  number  of  ophthal- 
mologists as  efficacious  in  the  treatment  of  pneumococcus  ulcers  of  the 
conjunctiva  and  cornea,  though  somewhat  irritating.  Zentmayer9  has 
recently  reported  very  favorably  upon  the  treatment  of  pneumococcus 
infections  of  the  eye,  and  particularly  corneal  ulcers  with  daily  applica- 


1  Ztschr.  f.  Immunitatsf.,  1912,  15,  625. 

2  Centralbl.  f.  Bakteriol.,  R.,  1912,  54,  6 


3  Berl.  klin.  Wchnschr.,  1912,  49,  2486. 

4  On  Pharmacotherapy  and  Preventive  Inoculation  Applied  to  Pneumonia  in  the 
African  Native,  1915.     (This  little  book  gives  a  complete  account  of  the  investiga- 
tions of  the  author  and  his  associates  with  ethylhydrocuprein  and  vaccines  in  human 
pneumococcus  infections.) 

5  Jour.  Exper.  Med.,  1915,  22,  551. 

6  Jour.  Exper.  Med.,  1915,  22,  269. 

7  Ztschr.  f.  Chemotherap.,  R.,  1912,  1,  1149. 

8  Jour.  Infect.  Dis.,  1917,  20,  272-347. 

9  Penna.  Med.  Jour.,  1917,  20,  487. 


CHEMOTHERAPY    IN    MALIGNANT    DISEASE  887 

tions  of  a  2  per  cent,  solution  of  the  hydrochloric!  and  the  hourly  instil- 
lation of  a  few  drops  of  a  1  per  cent,  solution  into  the  conjunctival  sac. 
In  the  treatment  of  lobar  pneumonia  the  consensus  of  opinion  would 
seem  to  indicate  that  the  drug  is  not  of  proved  value,  and  particularly 
must  care  be  exercised  in  the  dosage  to  avoid  the  amblyopia  which 
has  been  reported  by  a  number  of  investigators.  The  general  statement 
is  made  that  there  is  but  "a  small  interval  between  the  therapeutic  and 
the  toxic  dose." 

Tuberculosis. — Koga1  and  Otani2  have  recently  reported  favorably 
upon  the  treatment  of  tuberculosis  in  persons  and  experimental  animals 
with  a  substance  called  cyanocuprol.  Takano3  observed  beneficial 
results  in  the  treatment  of  6  cases  of  leprosy  with  the  same  substance. 
Lewis4  has  worked  extensively  with  new  synthetic  compounds  largely 
by  tests  in  vitro;  De  Witt,5  in  summarizing  the  results  of  chemothera- 
peutic  studies  in  tuberculosis,  concludes  that  the  results  so  far  with 
compounds  of  iodin,  arsenic,  copper,  gold,  mercury,  and  most  of  the 
dyes  have  been  in  the  main  negative. 

It  is  to  be  hoped,  therefore,  that  further  researches  will  result  in  the 
discovery  of  substances  that  have  a  marked  bactericidal  action  and 
that  are  yet  but  slightly,  if  at  all,  toxic  for  the  body  cells,  that  is,  sub- 
stances in  which  bacteriotropism  greatly  exceeds  organotropism.  It 
would  appear  that  this  discovery  is  possible  and  probable,  but  it  can 
be  accomplished  only  as  the  result  of  persistent  and  prolonged  research. 
Probably  the  most  wonderful  discovery  possible  in  medicine  would  be  a 
specific  remedy  for  tuberculosis,  and  this  may  be  within  the  realms  of 
chemotherapy. 

CHEMOTHERAPY  IN  MALIGNANT  DISEASE 

Brief  mention  may  be  made  of  certain  recent  advances  that  have 
been  made  in  the  experimental  chemotherapy  of  cancer  in  the  rat  and 
mouse.  While  the  pathogenesis  of  malignant  disease  is  still  unknown, 
specific  therapeutic  measures  of  this  kind  have  been  undertaken  on  the 
assumption  that  the  cancer-cell  is  different  from  the  normal  cell  from 
which  it  originated,  and  that  certain  substances  will  show  a  selective 
affinity  for  them;  in  other  words,  that  specificity  may  be  present  among 

1  Jour.  Exper.  Med.,  1916,  xxiv,  107  and  149. 

2  Jour.  Exper.  Med.,  1916,  xxiv,  187. 

3  Jour.  Exper.  Med.,  1916,  xxiv,  207. 

4  Bull.  Johns  Hopkins  Hosp.,  1917,  xxviii,  120. 

5  Jour.  Lab.  and  Clin.  Med.,  1916,  1,  677. 


888  CHEMOTHERAPY 

organotropic  substances.  Here,  of  course,  the  difficulties  are  great, 
because  the  structural,  biologic,  and  functional  differences  between  the 
normal  cell  and  the  cancer  cell  may  be  slight,  and  substances  must  be 
found  that  possess  a  high  affinity  for  the  cancer-cell  only. 

That  this  condition  may  indeed  exist  has  been  indicated  by  the  experi- 
ments of  Wassermann  and  his  collaborators.  These  investigations 
were  based  upon  the  discovery,  by  Gosio,  that  sodium  selenate  and  so- 
dium tellurate  are  more  rapidly  reduced  by  cancer-cells  than  by  normal 
cells,  and  that  this  reduction  takes  place  within  the  bodies  of  the  cells. 
Experiments  on  mouse  tumors  showed  that  the  injection  of  these  sub- 
stances into  the  growths  may  actually  lead  to  their  destruction,  the 
explanation,  according  to  Neuberg  and  Caspani,  being  that  certain 
compounds  of  the  heavy  metals  in  colloidal  form  favor  self-digestion 
(autolysis)  of  the  tumor  cells. 

The  problem  now  resolved  itself  into  finding  some  substance  that 
would  carry  the  metals  to  the  tumors,  or,  as  Wassermann  said,  "the 
building  of  rails  which  would  reach  the  tumor  and  by  which  the  selenium 
could  travel,"  as  local  injection  was  obviously  out  of  the  question  from 
the  practical  standpoint.  Eosin,  being  a  substance  endowed  with  great 
powers  of  diffusion,  was  selected  for  the  purpose,  and  a  number  of  eosin- 
selenium  compounds  were  tried  out.  The  results  were  encouraging,  as 
in  a  number  of  instances  the  tumors  became  soft  and  sloughed  away  or 
their  further  growth  was  checked.  "If  three  consecutive  daily  intrave- 
nous injections  of  the  eosin-selenium  compound  are  given  in  2.5  gin.  doses 
for  15-gram  mice,  a  distinct  softening  and  elasticity  of  the  tumor  are 
noticed  on  the  fourth  day;  on  the  fifth  day  a  fourth  injection  of  the 
same  dose  is  given,  after  which  there  is  no  longer  the  feeling  of  a  solid 
tumor,  but  rather  that  of  a  fluctuating  cyst  in  which  small,  movable 
tumor  particles  can  be  discovered.  After  the  fifth  injection  on  the 
seventh  day  this  soft  mass  becomes  smaller,  the  capsule  becomes  lax, 
and  the  configuration  of  a  circumscribed  tumor  can  no  longer  be  dis- 
tinguished, but  only  a  long  edematous  cord  can  be  felt.  Usually,  as  a 
result  of  the  sixth  injection,  in  favorable  cases,  the  absorption  and 
diminution  proceed  so  that  one  gets  the  feeling  of  an  empty  sac.  In 
ease  no  intercurrent  disease  occurs,  the  animal  is  cured  in  about  ten 
days,  with  a  disappearance  of  all  remnants  of  the  tumor." 

As  these  results  were  observed  after  intravenous  injection  of  the 
mixtures,  and  as  no  injury  to  the  body-cells  was  apparent,  it  seems  that  a 
step  forward  has  really  been  taken;  at  least,  these  investigators  have 
shown  that  it  is  possible  for  chemical  substances  to  pass  from  the  blood 
and  attack  tumor  cells.  Copper  and  tin  have  been  found  to  possess  a 


CHEMOTHERAPY    IN   MALIGNANT   DISEASE  889 

more  marked  affinity  for  tumor  cells,  and  the  whole  subject  is  probably 
just  at  the  threshold  of  further  discoveries  that  may  be  applied  with 
great  benefit  to  the  treatment  of  human  malignant  disease.  In  a  recent 
review  of  the  whole  subject  Weil1  has  concluded  that  when  the  results 
are  viewed  and  studied  in  a  critical  manner,  it  is  apparent  that  none  of  the 
various  methods  of  treatment  advocated  so  far  can  lay  claim  to  thera- 
peutic effectiveness.  According  to  Weil,  the  demonstrable  reduction 
in  the  size  of  a  tumor,  of  a  kind  not  to  be  attributed  to  the  natural 
processes  of  evolution  of  that  tumor  or  of  its  associated  lesions,  is  the 
one  essential  feature  of  effective  therapeutic  intervention. 

1  Jour.  Amer.  Med.  Assoc.,  1915,  64,  1283. 


PART  V 

INFECTION  AND  IMMUNITY 

INTRODUCTORY 

Methods. — The  exercises  and  experiments  herein  outlined  are  for 
the  purpose  of  teaching  the  principles  of  infection  and  immunity  by 
actual  laboratory  work,  whereby  the  student  performs  the  experiments 
and  is  taught  to  observe  the  results.  At  the  same  time  a  knowledge 
of  the  technic  of  immunologic  methods  is  obtained.  The  instructor  in 
charge  of  such  an  experimental  course  may  choose  certain  experiments 
in  outlining  a  course  according  to  the  allotment  of  time  and  purpose  of 
the  instruction. 

In  all  instances  I  have  outlined  the  experiments  according  to  average 
conditions.  It  is  readily  understood  that  differences  in  the  virulence  of 
a  certain  culture  or  the  weight  and  physical  condition  of  an  experimental 
animal  will  require  that  the  doses  advised  be  changed  to  meet  the  condi- 
tions. 

As  a  general  rule,  a  course  should  be  concentrated,  with  exercises  at 
least  three  times  a  week  in  order  that  the  student  may  follow  his  work 
closely  and  have  an  opportunity  for  making  adequate  and  accurate  ob- 
servations. 

AJI  attempt  is  made  to  bring  out  the  important  points  of  an  experi- 
ment by  a  few  pertinent  questions.  It  should  be  impressed  upon  the 
student  that  the  mind  should  be  held  open  for  observation  and  that  un- 
expected and  untoward  results  may  be  obtained  which,  however,  are 
always  of  interest  and  always  instructive  when  the  experiment  is  con- 
ducted in  a  careful,  methodical,  and  conscientious  manner.  Frequent 
general  discussions  should  be  held  for  a  general  review  of  the  subject  and 
correlation  of  facts  and  observations.  In  my  experience  students  are 
eager  for  the  work  and  seldom  fail  to  suggest  additional  work  in  the 
nature  of  original  research. 

The  Student. — 1.  The  student  should  work  protected  by  an  apron 
or  gown  with  short  sleeves;  he  should  be  careful  of  the  hands  and  avoid 
abrasions  and  cuts  and  carefully  wash  and  disinfect  the  hands  after  the 
work  of  each  day  has  been  completed. 

890 


ACTIVE    IMMUNIZATION    OF   ANIMALS  891 

2.  The  working  table  should  be  set  in  order  after  each  day's  work; 
pipets  and  soiled  glassware  should  be  properly  disposed  of,  instruments 
thoroughly  cleansed,  and  the  table  wiped  off  with  1  per  cent,  formalin 
solution.     It  should  be  impressed  upon  the  student  that  good  and  ac- 
curate work  is  seldom  done  amid  disorderly  and  dirty  surroundings. 

3.  The  student  must  never  sacrifice  accuracy  for  speed;  painstaking 
and  accurate  work  is  always  to  be  desired;    speed  is  gained  only  with 
practice. 

Records. — Each  worker  should  record  in  writing  in  a  suitable  note- 
book his  observations  of  the  various  tests  and  experiments.  Not  infre- 
quently unexpected  results  are  obtained,  and  it  is  important  to  under- 
stand and  explain  these  as  correctly  as  possible.  Note-books  should  be 
subject  to  frequent  inspection;  it  is  not  necessary  to  write  out  a  descrip- 
tion of  the  technic,  but  the  results  of  the  experiment  and  answers  to  the 
questions  should  be  set  forth  clearly  and  with  sufficient  detail. 

Animal  Experiments  and  Autopsies. — In  all  experiments  calling  for 
operative  procedure  an  anesthetic  is  to  be  used  in  order  that  unnecessary 
pain  be  avoided.  Ordinarily,  ether  is  to  be  employed,  or  in  rabbits  the 
rectal  injection  of  0.5  to  1  gram  of  chloral  hydrate  dissolved  in  5  to  10  c.c. 
of  water.  Autopsies  are  to  be  carefully  conducted,  and  the  lesions 
described  in  writing.  After  autopsy  the  table  is  to  be  scrubbed  and 
cleansed  with  a  solution  of  formalin,  and  the  carcass  disposed  of  by  in- 
cineration or  placing  in  a  solution  of  formalin  until  removed  and  other- 
wise disposed  of. 


EXERCISE  J.— ACTIVE  IMMUNIZATION  OF  ANIMALS 

EXPERIMENT  1. — PRODUCTION  OF  AGGLUTININS,  BACTERIOLYSINS,  AND 
OPSONINS 

1.  Secure  a  culture  of  Bacillus  typhosus,  an  old  laboratory  culture  of  cholera 
bacilli,  and  a  culture  of  Staphylococcus  aureus. 

2.  Proceed  to  immunize  two  rabbits  with  each  culture  by  intravenous  injections 
according  to  the  technic  described  on  page  68. 

3.  One  week  after  the  last  injection  the  animals  are  to  be  bled  and  the  serums 
secured  after  the  first  method  described  on  page  41. 

(a)  Define  the  meaning  of  antigen. 

(b)  What  are  antibodies? 

(c)  What  is  meant  by  active  immunization? 

(d)  What  precautions  should  be  observed  in  handling  these  antigens? 

(e)  What  precautions  are  to  be  observed  in  giving  intravenous  injec- 
tions? 


892  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

(f)  What  antibodies  do  you  suspect  are  present  in  these  immune 
serums? 

EXPERIMENT  2. — PRODUCTION  OF  PRECIPITINS 

1.  Immunize  a  rabbit  with  sterile  horse  serum  by  intravenous  injections  after  the 
first  method  as  described  on  page  70;    immunize  a  second  rabbit  after  the  second 
method. 

2.  Immunize  two  rabbits  with  sterile  human  serum  after  the  first  method. 

3.  Immunize  two  rabbits  with  cow  milk  by  intravenous  injections  after  the  third 
method. 

4.  Bleed  the  animals  from  the  carotid  artery  one  week  after  the  last  injection  and 
preserve  the  serums  in  a  sterile  condition. 

EXPERIMENT  3. — PRODUCTION  OF  HEMOLYSINS 

1.  Immunize  a  rabbit  with  washed  sheep  corpuscles  by  intravenous  injections 
after  the  first  method  as  described  on  page  72. 

2.  Immunize  a  second  rabbit  with  sheep  cells  after  the  second  method. 

3.  Immunize  a  third  rabbit  with  washed  human  cells  after  the  third  method. 

4.  Immunize  a  fourth  rabbit  with  washed  human  cells  by  intraperitoneal  in- 
jections as  described  on  page  73. 

5.  Bleed  the  animals  hi  four  days  to  a  week  after  the  last  injection  and  separate 
the  serums. 

6.  Prepare  a  portion  of  human  amboceptor  dried  on  paper  as  described  on  page 
71. 

7.  Preserve  the  balance  of  the  serum  and  the  other  serums  with  equal  parts  of 
neutral  glycerin. 

EXPERIMENT  4. — PRODUCTION  OF  CYTOTOXIN 

1.  Immunize  two  rabbits  with  dog  kidney  after  the  method  described  on  page  74. 

2.  After  the  immunization  has  been  completed,  preserve  the  serum  with  0.2  per 
cent,  phenol  in  sterile  ampules. 

While  these  various  immune  serums  are  being  prepared  the  student  is 
engaged  with  the  work  on  infection,  or  if  the  subject  of  immunity  is  taken 
up  at  once,  immune  serums  should  be  furnished  by  the  instructor. 


EXERCISE  2.— INFECTION 

EXPERIMENT  5. — EXPERIMENTAL  PNEUMONIA 

1.  Grow  a  virulent  culture  of  pneumococcus  in  glucose  serum  broth  for  forty- 
eight  to  seventy-two  hours  at  37°  C.  until  a  good  rich  growth  is  secured.     Prepare 
and  stain  smears  by  Gram's  method. 

2.  Pass  a  large  catheter  which  has  its  tip  cut  off  into  the  trachea  of  a  dog  until  it 
has  passed  into  one  of  the  primary  bronchi.     By  means  of  a  syringe  inject  quickly 
15  c.c.  of  culture;  remove  the  catheter  and  mouth-gag.     Take  the  rectal  temperature 
and  leukocyte  count  previous  to  inoculating. 

3.  Inject  a  second  dog  in  the  same  manner  with  2  c.c.  of  culture. 

4.  Inject  a  third  dog  with  5  c.c.  of  culture  intravenously. 


TOXINS  893 

5.  Observe  all  animals  for  forty-eight  hours,  taking  the  rectal  temperature  night 
and  morning.     Make  leukocyte  counts  every  four  hours  during  the  day.     Make 
physical  examination  of  the  chest. 

6.  Autopsy  the  animals  under  aseptic  precautions  and  with  complete  anesthesia. 

7.  Culture  the  heart's  blood  of  each  in  serum  bouillon. 

8.  Culture  the  pulmonary  lesions  in  serum  bouillon. 

9.  Prepare  smears  of  the  heart's  blood  and  lesions  and  stain  with  methylene-blue 
and  Gram's  stain. 

10.  Remove  consolidated  portions  of  lung  and  place  in  5  per  cent,  formalin. 
After  twenty-four  hours,  cut  sections  which  are  passed  through  by  the  paraffin 
method  and  stained  with  hematoxylin  and  eosin,  methylene-blue,  and  Gram's  stain. 

(a)  Did  any  of  the  animals  show  evidences  of  infection? 

(b)  Are  there  evidences  of  pneumonia?     How  do  these  lesions  com- 
pare with  those  of  human  pneumonia? 

(c)  How  do  you  explain  their  production? 

(d)  Does  the  animal  receiving  the  smaller  dose  of  pneumococci  intra- 
bronchially  show  evidences  of  pneumonia?     If  not,  why  not?     Does 
this  show  a  numerical  relationship  of  bacteria  to  infection? 

(e)  Were  the   temperature   changes   similar  to  those  observed  in 
human  lobar  pneumonia? 

(f)  Did  leukocytosis  occur  and  if  so,  why? 

(g)  Are  there  any  evidences  of  pleuritis  and  if  so,  how  do  you  explain 
its  production? 

(h)  Did  the  dog  receiving  the  pneumococci  intravenously  show  evi- 
dences of  pneumonia?  Does  this  bear  any  relation  to  the  question  of 
the  route  of  introduction  of  bacteria  to  infection? 

(i)  Are  the  pneumococci  seen  in  the  smears  of  the  lesions  and  blood 
encapsulated?  If  so,  what  is  the  significance  of  these  capsules?  Com- 
pare these  cocci  with  those  shown  in  the  smear  of  the  culture  before  in- 
jection? Are  the  capsules  lost  in  the  artificial  culture-media? 

(j)  Discuss  the  question  of  the  possible  modes  of  infection  in  human 
lobar  pneumonia,  especially  inspiratory  pneumonia. 


EXERCISE  3.— TOXINS 

EXPERIMENT  6. — DIPHTHERIA  TOXIN 

1.  Inoculate  tubes  of  neutral  or  slightly  alkaline  bouillon  with  a  virulent  culture 
of  diphtheria  bacilli  (Park-Williams  Bacillus  No.  8  being  especially  desirable). 

2.  Grow  at  35°  C.  for  five  days  and  filter  the  culture  through  a  Berkefeld  filter. 

3.  Inject  a  250-  to  300-gram  guinea-pig  in  the  median  abdominal  line  with  0.5 
c.c.  of  the  filtrate  (toxin). 

4.  Heat  1  c.c.  of  toxin  at  60°  C.  for  an  hour  and  inject  0.5  c.c.  into  a  second  guinea- 
pig  subcutaneously. 


894  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

5.  Observe  the  animals  for  at  least  four  days,  especially  for  the  development  of  a 
characteristic  edema  about  the  site  of  injection. 

6.  After  death  perform  a  careful  autopsy,  paying  particular  attention  to  the 
bloody  edema  at  the  site  of  injection  and  marked  hyperemia  of  the  suprarenal  glands. 
Make  cultures  on  Loeffler's  blood-serum  medium  of  edematous  area,  peritoneum,  and 
heart  blood. 

(a)  To  what  has  death  been  due? 

(b)  Has  diphtheria  toxin  a  selective  affinity  for  any  particular  tissue? 

(c)  Has  heat  any  effect  upon  diphtheria  toxin? 

(d)  Is  there  a  period  of  incubation  before  symptoms  develop  and  why? 

EXPEEIMENT  7. — METHOD  or  TESTING  THE  VIKULENCE  AND  TOXICITY 
OF  DIPHTHERIA  BACILLI 

1.  Make  a  culture  of  a  patient  harboring  the  bacilli  on  a  tube  of  Loeffler's  serum 
medium.     Inoculate  at  35°  C.  for  from  eighteen  to  twenty-four  hours;    prepare  a 
smear  and  stain  with  Loeffler's  methylene-blue.     If  diphtheria  bacilli  are  present, 
they  must  be  isolated  in  pure  culture.     Never  attempt  a  guinea-pig  test  with  an  impure 
culture. 

2.  Isolate  by  the  "streak"  method,  on  plates  of  blood-serum. 

3.  Inoculate  a  tube  of  1  per  cent,  glucose  bouillon,  which  is  neutral  or  slightly 
alkaline,  with  several  different  colonies. 

4.  Incubate  at  35°  C.  for  three  days,  keeping  the  tube  in  a  slanted  position  in 
order  to  give  the  culture  as  much  oxygen  as  possible.     If  a  good  growth  does  not 
appear  in  twenty-four  hours,  transplant  to  another  tube  of  bouillon  until  the  bacilli 
have  been  "educated"  to  grow  on  the  medium. 

5.  Examine  for  purity.     Select  a  250-  to  300-gram  guinea-pig  and  inject  2  c.c.  of 
the  unfiltered  culture  in  the  median  abdominal  line.     Annuals  over  the  weight  speci- 
fied are  more  resistant  and  less  reliable  for  test.     The  unfiltered  culture  is  used,  since 
toxin  is  but  one  element  of  the  disease-producing  power  of  diphtheria  bacilli,  and 
toxin  production  in  bouillon  may  not  be  a  true  index  of  the  toxin  production  in  mucous 
membranes. 

6.  Carefully  observe  the  animal  for  at  least  four  days.     Even  slight  toxemia, 
especially  if  accompanied  by  edema  at  the  site  of  injection,  should  be  regarded  as  a 
positive  result. 

7.  After  death  perform  a  careful  autopsy.     Make  cultures  of  the  edematous  area, 
peritoneum,  and  heart  blood.     Observe  whether  acute  hyperemia  of  the  suprarenal 
glands  is  present. 

8.  Not  infrequently  animals  showing  mild  or  even  an  absence  of  the  symptoms 
of  toxemia  develop  paralysis  of  the  hindquarters  two  or  three  weeks  later.  According 
to  Ehrlich,  this  paralysis  is  due  to  the  action  of  "toxon,"  a  toxic  substance  secreted, 
by  the  bacillus  or,  as  believed  by  others,  a  modified  form  of  toxin. 

9.  To  prove  that  diphtheria  was  the  cause  of  the  toxemia  or  death,  mix  2  c.c.  of 
the  culture  in  a  test-tube  with  1  c.c.  of  diphtheria   antitoxin  (500  units).     After 
standing  aside  for  an  hour  at  room  temperature,  inject  the  mixture  subcutaneously 
in  the  median  abdominal  line  of  a  250-  to  300-gram  guinea-pig. 

(a)  Is  the  diphtheria  bacillus  aggressive? 

(b)  What  evidence  have  you  that  the  lesions  and  death  are  due  to  a 
toxin? 


TOXINS  895 

(c)  Why  does  the  use  of  diphtheria  antitoxin  make  the  test  con- 
clusive? Would  tetanus  antitoxin  be  capable  of  neutralizing  diphtheria 
toxin? 

EXPERIMENT  8. — TETANUS  TOXIN 

Tetanus  toxin  is  composed  of  two  distinct  poisons  of  different  prop- 
erties. One,  tetanospasmin,  has  a  great  affinity  for  the  central  nervous 
system,  and  is  largely  responsible  for  the  symptoms  of  tetanus  infec- 
tion (neurotoxic) ;  the  second,  tetanolysin,  is  thermolabile  and  is  hema- 
toxic.  Tetanus  toxin  is  very  labile,  and  when  in  solution  soon  becomes 
attenuated.  For  these  experiments  it  is  necessary  to  use  either  fresh 
toxin  or  that  which  has  been  recently  precipitated  and  dried. 

1.  Secure  some  dried  tetanus  toxin  and  dissolve  in  sterile  salt  solution.     The 
toxin  may  be  secured  from  an  antitoxin  laboratory  and  preserved  indefinitely  in  the 
refrigerator.     Since  the  strength  varies  with  different  products,  the  lethal  dose  for  a 
300-gram  guinea-pig  should  be  ascertained  from  the  laboratory  furnishing  the  toxin. 

2.  Secure  2  grams  of  fresh  normal  guinea-pig  brain  and  liver.     Crush  in  separate 
sterile  mortars. 

3.  Add  a  lethal  dose  of  fresh  tetanus  toxin  and  5  c.c.  sterile  salt  solution  to  each. 
Mix  thoroughly  and  place  in  the  incubator  for  an  hour.     Remove  and  carefully  trans- 
fer to  sterile  centrifuge  tubes.     Centrifuge  thoroughly. 

4.  Inject   two  300-gram  guinea-pigs  subcutaneously  in  the  median  abdominal 
line  with  the  supernatant  fluids. 

5.  Inject  a  third  guinea-pig  with  a  similar  dose  of  toxin  and  5  c.c.  salt  solution 
(control). 

6.  Mark  pigs  carefully  and  observe  for  several  days. 

(a)  What  are  the  symptoms  of  tetanus? 

(b)  To  what  constituents  of  tetanus  toxin  are  the  chief  symptoms  due? 

(c)  Does  the  pig  which  received  the  toxin-brain  mixture  show  symp- 
toms? How  do  you  explain  the  result? 

(d)  Does  the  pig  which  received  the  toxin-liver  mixture  show  symp- 
toms? How  do  you  account  for  the  result? 

(e)  Is  the  tetanus  bacillus  characterized  by  its  toxicity  or  aggressive- 
ness? 

(f)  Is  there  a  period  of  incubation  before  symptoms  develop  and 
why? 

(g)  Autopsy  the  animal.     Are  there  any  definite  lesions  of  the  tissues 
and  internal  organs? 

EXPERIMENT  9. — TETANUS  TOXIN 

1.  Secure  some  fresh  tetanus  toxin. 

2.  Prepare  a  1  per  cent,  emulsion  of  washed  sheep  corpuscles  (washed  three  times 
in  an  excess  of  normal  salt  solution). 


896  EXPERIMENTAL   INFECTION    AND    IMMUNITY 

3.  Into  a  series  of  six  test-tubes  place  1  c.c.  of  the  corpuscle  emulsion  and  in- 
creasing doses  of  toxin:    0.1  c.c.,   0.2   c.c.,   0.4   c.c.,   0.8  c.c.,    1  c.c.,   2  c.c.     Add 
sufficient  normal  salt  solution  to  bring  the  total  volume  to  3  c.c. 

4.  Place  1  c.c.  of  the  corpuscle  suspension  in  2  c.c.  salt  solution  as  a  control  to 
make  sure  that  the  salt  solution  is  isotonic. 

5.  Heat  a  portion  of  toxin  at  60°  C.  for  an  hour  in  a  water-bath  and  set  up  a 
similar  set  of  tubes. 

6.  Incubate  all  tubes  for  two  hours  at  37°  C. 

7.  Read  results  at  the  end  of  this  time  and  again  after  the  tubes  have  settled  in 
the  refrigerator  overnight. 

(a)  Has  hemolysis  occurred  in  any  of  the  tubes? 

(b)  What  is  meant  by  hemolysis? 

(c)  What  constituent  of  tetanus  toxin  is  responsible  for  hemolyzing 
these  cells? 

(d)  Does  this  experiment  show  the  selective  affinity  of  a  toxin  for 
certain  cells? 

(e)  How  is  tetanus  toxin  produced? 

(f)  Does  heat  destroy  the  hemotoxic  agent? 


EXERCISE  4.— TOXINS  (Continued) 
EXPERIMENT  10. — BOTULISM  TOXIN 

1.  Prepare  toxin  by  cultivating  the  Bacillus  botulinus  in  an  alkaline  bouillon 
made  in  the  form  of  an  infusion  from  ham  with  the  addition  of  1  per  cent,  of  glucose, 
1  per  cent,  of  peptone,  and  1  per  cent,  of  sodium  chlorid.     Strict  anaerobic  cultures 
should  be  grown  for  four  weeks  and  filtered  through  a  Berkefeld  filter.     The  toxin 
may  be  preserved  in  brown,  sealed  vials,  and  kept  on  ice,  or  in  a  dried  form  in  vacuum. 

2.  Dilute  0.2  c.c.  of  the  toxin  with  3  or  4  c.c.  salt  solution  and  administer,  per  os, 
to  a  cat  by  means  of  a  small  catheter  passed  into  the  stomach. 

3.  Inject  0.1  c.c.  of  the  toxin  subcutaneously  in  the  abdominal  wall  of  a  rabbit. 

4.  Observe  animals  closely  for  several  hours  following  administration  of  the  toxin, 
for  some  products  are  so  highly  toxic  that  symptoms  may  appear  within  a  few  hours. 

(a)  What  are  the  symptoms  of  botulism  infection? 

(b)  Do  these  symptoms  show  any  selective  action  of  botulism  toxin 
for  certain  tissues? 

(c)  Autopsy  the  animals.     Are  there  any  gross  tissue  changes? 

EXPERIMENT  11. — DYSENTERY  TOXIN 

1.  Inoculate  a  flask  of  moderately  alkaline  bouillon  with  a  culture  of  dysentery 
bacilli,  preferably  of  the  Shiga-Kruse  type.     Inoculate  at  37°  C.  for  two  weeks  and 
pass  it  through  a  bacterial  filter. 

2.  Inoculate  three  rabbits  intravenously  with  1,  2,  and  5  c.c.  of  the  filtered  toxin. 

3.  Give  a  fourth  rabbit  2  c.c.  of  the  toxin  by  mouth  through  a  small  catheter 
passed  into  the  stomach. 

4.  Observe  the  animals  very  closely. 


TOXINS  897 

(a).  What  are  the  symptoms  of  dysentery  intoxication? 

(b)  Observe  the  temperature  at  frequent  intervals.     Are  there  any 
changes? 

(c)  Does  the  animal  which  received  the  toxin  by  mouth  show  symp- 
toms?    How  do  you  explain  the  result? 

(d)  Autopsy  the  animals  with  particular  attention  to  the  gastro- 
intestinal tract.     Are  there  any  evidences  of  a  selective  action  of  the 
toxin? 

EXPERIMENT  12. — STAPHYLOTOXIN 

Inject  2  c.c.  of  a  twenty-four-hour  bouillon  culture  of  Staphylococcus  aureus  in 
the  ear  vein  of  a  rabbit.  Take  the  temperature  before  and  every  twelve  hours 
after  injection.  Autopsy  the  animal  seventy-two  hours  later  under  aseptic  precau- 
tions. 

(a)  What  lesions  are  found? 

(b)  Where  are  these  chiefly  situated  and  why? 

(c)  Culture  the  heart's  blood  on  tubes  of  agar,  also  several  of  the 
lesions. 

(d)  After  twenty-four  hours'  incubation  examine  your  cultures. 

(e)  What  was  death  due  to? 

(f)  Define  bacteremia. 

(g)  Prepare  and  stain  sections  of  the  kidney,  including  a  lesion. 
What  are  the  histologic  changes?     How  do  you  explain  the  production 
of  these  tissue  changes? 

EXPERIMENT  13. — STAPHYLOTOXIN 

1.  Inoculate  a  flask  of  neutral  bouillon  with  a  virulent  culture  of  Staphylococcus 
aureus  and  grow  for  two  weeks  at  37°  C.     Filter  through  a  Berkefeld  filter. 

2.  Prepare  a  1  per  cent,  suspension  of  washed  rabbit  erythrocytes  in  normal  salt 
solution. 

3.  Into  a  series  of  six  test-tubes  place  1  c.c.  of  the  corpuscle  suspension  and  in- 
creasing amounts  of  Staphylococcus  filtrate:  .0.1  c.c.,  0.2  c.c.,  0.4  c.c.  0.8  c.c.,  1  c.c., 
and  2  c.c.     Add  normal  salt  solution  to  bring  the  total  volume  to  3  c.c. 

4.  Prepare  a  control  by  placing  1  c.c.  of  corpuscle  suspension  in  2  c.c.  salt  solution. 

5.  Incubate  tubes  for  two  hours  at  37°  C.  and  read  results. 

6.  This  test  will  be  referred  to  again  in  the  technic  for  determining  the  antilysin 
in  blood-serum. 

(a)  Has  hemolysis  occurred  in  any  of  the  tubes? 

(b)  To  what  constituent  of  the  filtrate  are  these  results  due? 

(c)  Does  this  experiment  show  the  selective  action  of  a  toxin? 

(d)  Do  the  results  have  any  bearing  upon  the  anemia  and  jaundice 
of  severe  Staphylococcus  infections? 

57 


EXPERIMENTAL   INFECTION   AND    IMMUNITY 


EXERCISE  5.— TOXINS  (Continued)— PLANT  AND  ANIMAL  TOXINS 
EXPERIMENT  14.— STREPTOTOXIN 

1.  Cultivate  a  strain  of  virulent  streptococci  in  tubes  of  slightly  alkaline  serum 
or  ascites  bouillon  for  forty-eight  hours  at  37°  C. 

2.  Inoculate  a  guinea-pig  intraperitoneally  with  1  or  2  c.c.  of  the  unfiltered  cul- 
ture. 

3.  Autopsy  aseptically  eighteen  or  twenty-four  hours  later. 

(a)  What  are  the  gross  features  of  the  exudate? 

(b)  Prepare  smears  of  the  exudate  and  stain  with  methylene-blue. 
Are  there  many  cells  present?     How  do  you  explain  the  cellular  con- 
tent? 

(c)  Are  streptococci  present?    Are  these  inclosed  by  any  of  the 
cells?     How  do  you  explain  the  condition? 

,(d)  Is  the  exudate  bloody?     If  so,  why? 

(e)  Examine    your    blood-agar    plates.     Are    there    any  peculiar 
changes  around  the  colonies?     To  what  are  these  changes  due?     Does 
this  have  any  connection  with  the  bloody  character  of  the  exudate? 

(f)  Why  are  streptococcus  infections  so  virulent  and  spreading  in 
character? 

(g)  Is  the  streptococcus  an  aggressive  microorganism? 
(h)  Do  streptococci  contain  an  endotoxin? 

EXPERIMENT  15. — PHYTOTOXINS 

1.  Secure  0.01  gm.  ricin  or  abrin  and  dissolve  in  10  c.c.  normal  salt  solution  or 
distilled  water. 

2.  Inject  1  c.c.  intravenously  into  a  rabbit.     Place  the  animal  in  a  metabolic 
cage  and  collect  urine,  or  catheterize  every  twelve  hours  or  at  death. 

3.  Examine  the  urine  for  hemoglobin  and  erythrocytes. 

4.  Prepare  a  1  per  cent,  suspension  of  washed  rabbit  and  guinea-pig  corpuscles. 

5.  Into  a  series  of  six  small  test-tubes  place  increasing  doses  of  the  ricin  or  abrin 
solution  as  follows:  0.1,  0.2,  0.3,  0.4,  0.5,  and  0.8  c.c.     Add  1  c.c.  of  rabbit-cell  emul- 
sion to  each  and  sufficient  normal  salt  solution  to  make  the  total  volume  in  each  tube 
equal  to  2  c.c.     A  seventh  tube  is  the  corpuscle  control  and  contains  1  c.c.  of  the  ery- 
throcyte  suspension  and  1  c.c.  of  salt  solution. 

6.  Prepare  a  similar  series  of  tubes  with  the  guinea-pig  erythrocyte  suspension. 

7.  Shake  the  tubes  gently  and  incubate  for  two  hours. 

(a)  Do  any  of  the  tubes  show  hemolysis  or  hemagglutination? 

(b)  Is  the  action  the  same  with  both  bloods? 

(c)  Does  the  plant  toxin  show  a  selective  affinity? 

(d)  Does  the  rabbit  show  any  evidences  of  hemolytic  jaundice? 
Are  there  blood  elements  in  the  urine? 


ENDOTOXINS   AND   AGGRESSINS  899 

(«)  Autopsy  the  animal,  paying  particular  attention  to  the  kidneys 
and  gastro-intestinal  tract?     What  changes  have  occurred? 

EXPERIMENT  16. — ZOOTOXIN  (COBRA  VENOM) 

1.  Collect  about  2  c.c.  of  normal  human  blood  and  place  in  5  c.c.  of  2  per  cent, 
sodium  citrate  in  0.85  per  cent,  sodium  chlorid  solution.     This  mixture  must  not  be 
shaken  and  the  cells  should  be  washed  at  least  four  times  with  normal  salt  solution, 
after  which  they  are  made  up  to  a  4  per  cent,  suspension. 

2.  Prepare  a  4  per  cent,  suspension  of  sheep  corpuscles  in  the  same  manner. 

3.  Prepare  a  stock  dilution  of  cobra  venom  by  dissolving  0.005  gm.  dried  venom 
in  10  c.c.  of  normal  salt  solution.     This  solution  will  keep  about  one  week  in  the  re- 
frigerator. 

4.  Prepare  a  solution  of  venom  1 : 10,000  by  placing  2  c.c.  of  the  stock  solution  in 
8  c.c.  salt  solution.     Prepare  a  T:  20,000  dilution  by  placing  2  c.c.  of  dilution  1 : 10,000 
in  2  c.c.  salt  solution. 

5.  Place  1  c.c.  of  each  corpuscle  suspension  into  two  small  test-tubes  and  add  1 
c.c.  and  0.5  c.c.  each  venom  dilution.     Shake  the  tubes  gently  and  place  in  the  incu- 
bator for  an  hour  and  then  in  the  refrigerator  overnight. 

6.  Inject  2  c.c.  of  the  1 : 10,000  dilution  intravenously  into  a  rabbit  and  1  c.c. 
intravenously  into  a  guinea-pig. 

7.  Observe  the  animals  closely,  particularly  the  urine. 

(a)  Does  the  venom  show  a  hemotoxic  action?     Does  it  act  the 
same  on  rabbit  and  guinea-pig  corpuscles?     If  not,  why  not? 

(b)  How  do  you  explain  venom  hemolysis? 

(c)  What  are  the  evidences  of  venom  hemolysis  in  vivo? 

(d)  Is  the  hemotoxic  poison  the  most  dangerous  constituent  of  venom? 


EXERCISE  6,— ENDOTOXINS  AND  AGGRESSINS 

EXPERIMENT  17. — ENDOTOXINS 

1.  Incubate  eight  agar  slants  with  Bacillus  typhosus  and  cultivate  at  37°  C.  for 
seventy-two  hours. 

2.  Wash  off  the  growths  with  5  c.c.  distilled  water  for  each  tube. 

3.  Place  the  emulsion  in  a  sterile  bottle  with  glass  beads  and  shake  for  twenty- 
four  hours  at  room  temperature.     At  the  same  tune  inoculate  six  more  slants  of  agar 
and  wash  off  the  growths  at  the  end  of  twenty-four  hours  with  3  c.c.  of  normal  salt 
solution  for  each  tube. 

4.  Filter  the  emulsion,  which  has  been  shaken,  through  a  Berkefeld  filter. 

5.  Inject  two  rabbits  intravenously  with  2  c.c.  each  of  the  filtrate  (endotoxins) 
and  unfiltered  culture. 

6.  .Observe  influence  on  body-weight  and  temperature  and  for  symptoms  of 
toxemia. 

7.  Inject  three  pigs  intraperitoneally  as  follows: 

1st  pig:  2  c.c.  of  emulsion  of  typhoid  bacilli. 

2d  pig:  2  c.c.  of  filtrate. 

3d  pig:  1  c.c.  each  of  emulsion  and  filtrate. 


900  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

(a)  Are  there  symptoms  of  toxemia  in  the  rabbits?     Are  these  symp- 
toms alike  in  both  animals?     Which  is  more  toxic,  the  nitrate  or  the 
whole  culture? 

(b)  Does  the  filtrate  enhance  the  effect  of  the  whole  culture? 

(c)  If  any  of  the  animals  succumb,  autopsy  under  aseptic  precau- 
tions.    Prepare  cultures  of  the  peritoneum  and  heart's  blood  on  agar 
slants  or  in  neutral  bouillon.     Is  the  typhoid  bacillus  markedly  ag- 
gressive? 

(d)  If  there  is  a  peritoneal  exudate,  prepare  smears  and  stain  with 
carbolfuchsin  (1  :10).     What  type  of  cell  predominates?     Are  any  of 
the  bacilli  engulfed  in  the  cells? 

(e)  Do  any  of  the  bacilli  show  signs  of  disintegration?     If  so,  do  you 
know  what  these  changes  are  due  to? 

EXPERIMENT  18. — NATURAL  AGGRES&INS 

1.  Inject  a  guinea-pig  intraperitoneally  with  3  c.c.  of  twenty-four-hour  bouillon 
culture  of  Bacillus  typhosus. 

2.  After  twenty-four  hours,  anesthetize  the  animal  and  remove  the  exudate 
aseptieally  into  5  c.c.  of  a  2  per  cent,  solution  of  sodium  citrate  to  prevent  coagula- 
tion. 

3.  Filter. 

4.  Inject  three  guinea-pigs  intraperitoneally  as  follows: 

1st  pig:  3  c.c.  of  filtrate. 

2d  pig:  2  c.c.   of  twenty-four-hour  bouillon  culture  of 

typhoid  bacilli. 
3d  pig:  1  c.c.  each  of  culture  and  filtrate. 

5.  Observe  animals  for  several  days,  particularly  the  temperature  reaction  and 
weights. 

(a)  Is  the  filtrate  alone  toxic? 

(b)  Does  the  filtrate  appear  to  enhance  the  effects  of  the  unfiltered 
culture? 

(c)  If  the  animals  succumb,  autopsy  aseptieally.     Make  cultures 
of  the  peritoneum  and  heart's  blood  on  agar  slants  or  in  bouillon.     Pre- 
pare smears  of  the  exudate  and  stain  with  1  : 10  carbolfuchsin.     Do  any 
of  the  animals  show  a  typhoid  bacteremia?     Does  the  filtrate  appear  to 
prevent  phagocytosis? 


EXERCISE  7.— BACTERIAL  PROTEIN;  PTOMAINS;  MECHANICAL  ACTION 

OF  BACTERIA 

EXPERIMENT  19. — BACTERIAL  PROTEIN  (VAUGHAN) 

1.  Inoculate  10  slants  of  2  per  cent,  neutral  agar  in  large  bottles  with  a  culture 
of  Bacillus  coli  and  grow  at  37°  C.  for  four  days. 

2.  Add  about  10  c.c.  of  distilled  water  to  each  bottle  and  gently  remove  the 


BACTERIAL   PROTEIN;     PTOMAINS  901 

growth  with  a  sterilized  platinum  wire,  being  particularly  careful  not  to  remove  frag- 
ments of  agar. 

3.  Pipet  the  heavy  emulsion  to  a  large  centrifuge  tube.     Another  cubic  centi- 
meter or  two  of  water  is  added  to  each  bottle  to  remove  the  balance  of  the  growth. 

4.  Centrifuge  at  high  speed  until  the  bacilli  are  thoroughly  settled.     Decant  off 
the  supernatant  fluid  and  add  50  per  cent,  alcohol;  mix  and  centrifuge.     Decant  and 
add  95  per  cent,  alcohol;  mix  and  centrifuge. 

5.  Remove  the  sediment  of  bacteria  to  a  small  flask  with  50  c.c.  of  absolute 
alcohol  and  set  aside  at  room  temperature  for  a  day.     Decant  off  the  alcohol  and  add 
50  c.c.  of  ether.     Mix  and  set  aside  for  another  twenty-four  hours.     Decant  off  the 
ether  that  remains  and  remove  sediment  to  a  porcelain  or  agate  mortar.     Place  in 
the  incubator  for  a  few  hours  until  thoroughly  dry. 

6.  Grind  the  dry  mass  very  thoroughly,  the  operator  wearing  a  mask,  until  a 
fine  powder  is  produced.     Place  the  powder  of  bacterial  substance  in  a  wide-mouthed 
dark-glass  bottle  and  preserve  in  a  dark  closet.     A  portion  will  be  needed  later  for 
experiments  in  anaphylaxis. 

7.  Mix  0.02  gm.  of  the  dry  powder  with  10  c.c.  salt  solution. 

8.  Inject  2  c.c.  of  this  emulsion  intraperitoneally  in  a  300-gram  guinea-pig. 

9.  Heat  2  c.c.  of  the  emulsion  at  60°  C.  for  two  hours  and  inject  intraperitoneally 
in  a  guinea-pig. 

10.  Inject  a  third  pig  intraperitoneally  with  2  c.c.  of  a  four-day  bouillon  culture 
of  Bacillus  coli. 

(a)  Could  endotoxins  withstand  these  various  manipulations? 

(b)  Does  the  heated  extract  kill  the  animal  more  quickly  than  the 
unheated,  and  if  so,  why? 

(c)  What  are  the  symptoms  produced? 

(d)  Autopsy  the  animals.     Are  there  evidences  of  peritonitis?    Are 
there  differences  in  the  lesions  of  the  three  animals?     If  so,  how  do  you 
explain  them? 

(e)  The  bacterial  split  portion  will  be  studied  later  under  Anaphylaxis. 


EXPERIMENT  20. — PTOMAINS 

1.  Procure  4  ounces  of  beef  and  mince. 

2.  Place  in  a  flask  with  250  c.c.  tap  water  and  inoculate  with  a  culture  of  Bacillus 
coli.     Fit  the  flask  with  a  rubber  stopper  with  a  glass  tube  to  carry  off  gases. 

3.  Inoculate  a  flask  of  neutral  bouillon  at  the  same  time  with  the  same  culture. 

4.  Cultivate  both  at  37°  C.  for  two  weeks. 

5.  Filter  both  through  a  coarse  Berkefeld  filter. 

6.  Concentrate  both  filtrates  to  one-half  their  volume  at  a  low  temperature  on  a 
water-bath. 

7.  Inject  two  guinea-pigs  with  each  preparation,  giving  1  c.c.  subcutaneously 
and  0.5  c.c.  intraperitoneally. 

8.  Observe  the  four  animals  closely. 

(a)  What  symptoms  develop  in  both  sets  of  animals? 

(b)  What  are  ptomains?    What  role  do  they  play  in  the  production 
of  disease? 

(c)  Can  antibodies  be  produced  for  true  ptomains? 


902  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

EXPERIMENT  21. — MECHANICAL  ACTION  OF  BACTERIA 

1.  Inject  a  300-gram  pig  intraperitoneally  with  2  c.c.  of  a  forty-eight-hour-old 
bouillon  culture  of  Bacillus  anthracis.     Great  care  must  be  exercised  in  handling  and 
injecting  this  culture. 

2.  Autopsy  the  animal  at  the  end  of  forty-eight  hours  and  make  cultures  on  agar 
slants  of  the  heart's  blood,  liver,  and  spleen.     Remove  the  heart,  lungs,  liver,  spleen, 
and  kidneys,  and  place  in  2  per  cent,  formalin.     Prepare  smears  of  the  blood  and 
stain  with  Gram's  method. 

3.  After  fixing  for  twenty-four  hours,  sections  of  these  organs  are  to  be  prepared 
and  stained  with  methylene-blue  and  with  Gram's  stain. 

4.  Examine  the  cultures  at  the  end  of  twenty-four  hours. 

5.  Examine  the  sections. 

(a)  Is  the  anthrax  bacillus  aggressive? 

(b)  Does  the  anthrax  bacillus  produce  much  toxin? 

(c)  Did  the  animal  show  any  symptoms  of  -its  infection? 

(d)  Are  there  evidences  of  peritonitis? 

(e)  How  do  you  explain  the  probable  causes  of  death  in  human  an- 
thrax? 


EXERCISE  8.— KINDS  OF  IMMUNITY 

EXPERIMENT  22. — PHAGOCYTOSIS  IN  NATURAL  IMMUNITY 

1.  Inject  a  healthy  guinea-pig  intraperitoneally  with  1  c.c.  of  a  twenty-four- 
hour  or  forty-eight-hour  glucose  bouillon  culture  of  streptococci  of  low  or  moderate 
virulence.     A  culture  of  staphylococci  may  be  used  instead,  although  the  former  is 
preferable. 

2.  At  the  end  of  eighteen  hours  study  the  peritoneal  fluid.     Prepare  smears  and 
stain  with  methylene-blue    or   other  suitable  stain.     Prepare  cultures  in  glucose 
bouillon  of  the  heart's  blood. 

(a)  Did  the  animal  show  any  evidences  of  infection? 

(b)  Has  phagocytosis  occurred  in  the  peritoneal  cavity? 

(c)  What  constituent  of  normal  serum  aids  phagocytosis? 

(d)  Did  a  bacteremia  develop? 

EXPERIMENT  23. — NATURAL  ANTIBACTERIAL  IMMUNITY 

1.  Inject  a  250-gram  guinea-pig  subcutaneously  with  1  c.c.  of  a  twenty-four-hour 
bouillon  culture  of  Bacillus  anthracis. 

2.  Inject  a  large  albino  rat  with  the  same  dose  and  in  the  same  manner. 

3.  Observe  the  animals  for  twenty-four  to  forty-eight  hours.     At  autopsy  pre- 
pare smears  and  cultures  of  the  heart  blood  of  each.    Stain  the  smears  with  methylene- 
blue  and  according  to  the  method  of  Gram. 

Do  the  animals  present  symptoms   of  infection?    Are  there  any 
differences  between  the  two? 


ACQUIRED    IMMUNITY  903 

EXPERIMENT  24. — RELATIVE  FACTORS  IN  NATURAL  IMMUNITY 

1.  Place  a  frog  in  a  shallow  vessel  of  warm  water  in  the  incubator  at  37°  C. 

2.  After  twenty-four  hours  give  a  subcutaneous  injection  of  tetanus  toxin  equal 
to  one-tenth  the  fatal  dose  for  a  300-gram  guinea-pig. 

3.  Inject  a  second  frog  with  an  equal  amount  of  toxin  and  keep  it  at  ordinary 
room  temperature  in  cold  water. 

4.  Observe  both  animals. 

(a)  Do  symptoms  of  tetanus  develop  in  any  of  the  animals? 

(b)  Why  does  heat  favor  tetanus  infection? 

(c)  Give  further  examples  of  relative  natural  immunity. 

(d)  Are  anthrax  bacilli  found  in  the  .smears  and  cultures  of  both  ani- 
mals?    Which  animal  is  immune?     Could  phagocytosis  play  a  role  in 
J;his  immunity? 

EXPERIMENT  25. — INFLUENCE  OF  TEMPERATURE  UPON  NATURAL  IM- 
MUNITY 

1.  Procure  dried  tetanus  toxin  and  dissolve  sufficient  in  6  c.c.  normal  salt  solu- 
tion equivalent  to  6  lethal  doses  for  a  350-gram  guinea-pig. 

2.  Inject  2  c.c.  into  the  pectoral  muscles  of  a  young  hen.     Take  the  rectal  tem- 
perature. 

3.  Inject  2  c.c.  into  a  second  hen.     Take  the  rectal  temperature  and  place  her 
in  cold  water  until  the  temperature  has  dropped  several  degrees. 

4.  Place  hen  No.  1  in  a  cage  and  keep  her  at  ordinary  laboratory  temperature. 

5.  Place  hen  No.  2  in  a  cage  and  keep  her  in  a  cold  place  or  renew  the  bath 
several  times  in  order  to  keep  her  temperature  subnormal. 

(a)  What  is  the  normal  temperature  of  the  hen? 

(b)  Do  both  animals  present  symptoms  of  tetanus? 

(c)  How  do  you  explain  the  difference? 

6.  If  hen  No.  1  does  not  show  symptoms  of  tetanus  by  the  second  day,  reinject 
her  with  an  amount  of  toxin  equal  to  10  lethal  doses  for  a  guinea-pig. 

(a)  Does  this  hen  present  evidences  of  tetanus? 

(b)  If  so,  how  do  you  explain  the  result? 


EXERCISE  9.— ACQUIRED  IMMUNITY 

EXPERIMENT  26. — ACQUIRED  ACTIVE  (ANTIBACTERIAL)  IMMUNITY 

1.  Immunize  a  rabbit  with  typhoid  bacilli  as  described  in  the  chapter  on  Active 
Immuniz  at  ion . 

2.  Inject  this  immune  rabbit  intravenously  with  6  loopfuls  of  a  twenty-four- 
hour  culture  of  Bacillus  typhosus  thoroughly  emulsified  in  2  c.c.  sterile  salt  solution. 

3.  Inject  a  normal  rabbit  of  equal  weight  with  an  equal  dose  and  in  the  same 
manner. 

4.  Both  animals  are  weighed  and  their  temperatures  recorded  before  the  injec- 


904  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

tions  are  given.  If  possible,  take  temperature  every  four  hours.  Observe  both 
animals  for  symptoms  of  infection.  If  one  or  both  succumb,  autopsy  aseptically  and 
culture  the  heart  blood  in  neutral  bouillon  or  bile. 

(a)  Are  there  differences  in  the  clinical  condition  of  the  two  animals? 

(b)  To  what  do  you  ascribe  these  differences? 

(c)  What  chief  antibody  is  responsible  for  the  destruction  of  the 
bacilli  in  the  immune  animal? 

(d)  Has  typhoid  bacteremia  developed? 

PASSIVE  ACQUIRED  IMMUNITY 
EXPERIMENT  27. — ACQUIRED  PASSIVE  (ANTITOXIC)  IMMUNITY 

1.  Inject  a  250-  to  300-gram  guinea-pig  subcutaneously  with  1  c.c.  (500  units)  of 
diphtheria  antitoxin  (pig  No.  1). 

2.  Secure  diphtheria  toxin  in  which  the  lethal  dose  (L.  D.)  for  a  250-  to  300-gram 
guinea-pig  is  known. 

3.  Place  this  dose  of  toxin  in  a  small  test-tube  or,  better,  in  the  barrel  of  a  pre- 
cision syringe  (Hitchens),  and  add  1  c.c.  antitoxin  (500  units).     Mix  thoroughly  and 
keep  at  room  temperature  for  an  hour. 

4.  Inject  pig  No.  1  with  an  amount  of  toxin  equal  to  the  L.  D.  dose. 

5.  Inject  the  toxin  and  antitoxin  mixture  into  a  second  pig  of  250  to  300  grams. 

6.  Inject  the  same  amount  of  toxin  into  a  third  pig  of  equal  weight  as  a  control. 

7.  Observe  animals  closely  for  at  least  four  or  five  days. 

(a)  What  are  the  chief  symptoms  of  diphtheria  intoxication  of  the 
guinea-pig? 

(b)  Do  all  animals  show  these  symptoms? 

(c)  How  do  you  explain  the  absence  of  symptoms  in  pig  No.  1? 
How  in  pig  No.  2  ?     Is  the  mechanism  of  protection  the  same  in  both? 

(d)  What  bearing  has  this  experiment  upon  the  treatment  of  diph- 
theria? 

EXPERIMENT  28. — ACQUIRED  PASSIVE  (ANTITOXIC)  IMMUNITY 

The  above  experiment  may  be  conducted  in  exactly  the  same  manner,  using 
tetanus  toxin  and  antitoxin. 

EXPERIMENT  29. — ACQUIRED  PASSIVE  (ANTIBACTERIAL)  IMMUNITY 

1.  Secure  a  culture  of  pneumococcus,  and  if  its  lethal  dose  for  white  mice  is  un- 
known, determine  this  by  injecting  a  series  of  mice  with  decreasing  doses  of  a  forty- 
eight-hour  serum  bouillon  culture. 

2.  Secure  a  few  cubic  centimeters  of  antipneumococcus  serum,  especially  a  serum 
prepared  with  the  culture  being  used,  or  at  least  one  that  is  polyvalent. 

3.  Inject  three  mice  subcutaneously  with  0.1,  0.5,  and  1  c.c.  of  the  serum.     Then 
inject  them  subcutaneously  with  double  the  lethal  dose  of  pneumococci.     Inject  a 
fourth  mouse  with  culture  alone  (control). 

4.  Observe  the  animals  for  several  days,  and  at  autopsy  culture  the  heart  blood  in 
tubes  of  serum  bouillon  and  prepare  smears  which  are  to  be  stained  according  to 
Gram. 


PHAGOCYTOSIS  905 

(a)  Has  the  serum  served  to  protect  any  of  the  mice? 

(b)  Is  this  protection  due  to  bacteriolysins,  bacteriotropins,  or  both? 
Do  antitoxins  play  any  part? 

(c)  Why  is  it  preferable  to  use  homologous  culture  and  immune 
serum?     What  bearing  has  this  upon  the  treatment  of  human  pneumo- 
coccus  infections? 

(d)  Do  you  know  a  method  for  quickly  isolating  and  identifying 
pneumococci  from  sputum? 

Note. — This  experiment  may  be  conducted  with  rabbits;   also  a  cul- 
ture of  streptococcus  and  its  immune  serum  may  be  used. 


EXERCISE  JO.— PHAGOCYTOSIS 

EXPERIMENT  30. — PHAGOCYTOSIS  (MACROPHAGES) 

1.  Secure  pigeons'  blood  and  defibrinate.     Wash  the  corpuscles  several  times  and 
prepare  a  5  per  cent,  suspension. 

2.  Inject  a  guinea-pig  intraperitoneally  with  3  c.c.  of  this  corpuscle  suspension. 

3.  After  three  hours  withdraw  a  small  amount  of  peritoneal  exudate  by  means 
of  a  capillary  pipet.     Examine  with  hanging-drop  preparations  and  prepare  smears 
and  stain  with  Wright's  stain. 

4.  Make  similar  preparations  twelve,   eighteen,   twenty-four,   and  forty-eight 
hours  after  injection. 

(a)  Has  phagocytosis  occurred? 

(b)  Which  cells  have  become  phagocytes? 

(c)  Do  the  pigeon  cells  appear  as  if  undergoing  digestion? 

(d)  Which  portion  of  the  pigeon  cell  resists  digestion? 

(e)  Explain  mechanism  of  intracellular  digestion. 

EXPERIMENT  31. — PHAGOCYTOSIS  (MICROPHAGES) 

1.  Place  a  drop  of  blood  in  the  hollow  cell  of  a  ground-out  slide  such  as  is  used  for 
hanging-drop  preparations.     Cover  with  a  clean  slide,  seal  with  a  ring  of  vaselin,  and 
place  in  a  large  Petri  dish  containing  pieces  of  filter-paper  moistened  with  water 
(moist  chamber).     Place  in  the  incubator  for  fifteen  minutes. 

2.  Remove  the  cover-glass  and  carefully  wash  cover-glass  and  cell  with  normal 
salt  solution.     The  erythrocytes  are  removed  and  the  leukocytes  left  adherent  to  the 
cell  and  cover-glass. 

3.  Fill  the  cell  with  fresh  serum  and  add  a  quantity  of  culture,  preferably  a  loop- 
ful  of  a  twenty-four-hour  bouillon  culture  of  non-virulent  anthrax  bacilli.     Apply  the 
cover-glass  and  vaselin  the  margins  to  prevent  evaporation. 

4.  If  at  all  possible,  employ  a  warm  stage  and  watch  the  process  under  the  micro- 
scope. 

(a)  Does  phagocytosis  occur? 

(b)  Which  cells  are  acting  as  phagocytes? 


906  EXPERIMENTAL   INFECTION  AND    IMMUNITY 

(c)  Can  you  make  out  the  phase  of  fixation  and  phase  of  ingestion? 

(d)  By  what  agencies  do  leukocytes  kill  engulfed  bacteria? 

EXPERIMENT  32. — PHAGOCYTOSIS 

1.  Inject  a  guinea-pig  intraperitoneally  with  5  c.c.  of  finely  divided  cinnabar 
suspension  and  1  c.c.  subcutaneously  on  each  side  in  the  region  of  the  inguinal  lymph- 
glands. 

2.  Autopsy  the  animal  at  the  end  of  twenty-four  hours.     Prepare  hanging-drop 
preparations  and  smears  of  the  peritoneal  exudate  (stained  lightly  with  methylene- 
blue).     Prepare  sections  of  the  inguinal  and  abdominal  lymphatic  glands. 

(a)  Has  phagocytosis  occurred  in  the  peritoneal  cavity?     Which 
cells  have  become  phagocytic? 

(b)  Do  you  find  phagocytes  in  the  lymph-glands?    Where  are  the 
leukocytes  situated?     Which  cells  have  become  phagocytes? 

(c)  How  did  the  material  reach  the  glands? 

(d)  How  do  you  explain  the  presence  of  cinnabar  in  the  endothelial 
cells  of  the  gland? 


EXERCISE  H.— CHEMOTAXIS 
EXPERIMENT  33. — POSITIVE  CHEMOTAXIS 

1.  Inject  a  guinea-pig  intraperitoneally  with  1  or  2  c.c.  of  a  twenty-four-hour 
culture  of  Staphylococcus  aureus. 

2.  Autopsy  the  animal  eighteen  hours  -later  and  prepare  smears  of  the  exudate. 
Fix  with  methyl  alcohol  for  five  minutes,  dry,  and  stain  with  carbol-thionin,  Wright's 
or  Loeffler's  methylene-blue,  counterstained  with  eosin. 

3.  Prepare  cultures  of  the  peritoneal  exudate. 

(a)  Describe  the  exudate. 

(b)  Has  phagocytosis  occurred? 

(c)  Which  cells  are  actively  phagocytic? 

(d)  Are  all  the  cocci  engulfed? 

(e)  Are  there  any  evidences  of  the  cocci  undergoing  intracellular 
digestion? 

(f)  Could  a  sterile  substance  call  forth  an  exudation  of  leukocytes 
when  injected  into  a  serous  cavity? 

EXPERIMENT  34. — NEGATIVE  CHEMOTAXIS 

1.  Inject  a  guinea-pig  intraperitoneally  with  0.5  to  1  c.c.  of  a  forty-eight-hour 
serum  bouillon  culture  of  virulent  streptococci. 

2.  Autopsy  at  the  end  of  eighteen  to  twenty-four  hours  if  death  has  not  already 
occurred. 

3.  Prepare  cultures  in  serum  bouillon  and  a  number  of  smears.     Stain  the  latter 
with  methylene-blue  or  according  to  Gram. 


OPSONINS  907 

(a)  Describe  the  exudate.     How  does  it  differ  from  that  found  in 
the  preceding  experiment? 

(b)  How  do  you  explain  the  serous  character  of  the  exudate? 

(c)  Has  phagocytosis  occurred? 

(d)  Do  you  think  the  streptococci  have  multiplied  in  the  peritoneal 
cavity? 

(e)  What  means  could  you  suggest  for  overcoming  this  action  of 
virulent  streptococci? 


EXERCISE  12.— OPSONINS 

EXPERIMENT  35. — NORMAL  OPSONINS 

1.  Prick  the  finger  and  secure  1  c.c.  of  blood  in  a  small  test-tube.     Also  1  c.c.  in  a 
centrifuge  tube  containing  2  c.c.  of  sodium  citrate  solution.     After  coagulation  re- 
move the  serum  from  the  first  tube. 

2.  Divide  the  serum  into  two  portions  and  heat  one  portion  at  56°  C.  for  thirty 
minutes. 

3.  Prepare  an  emulsion  of  leukocytes  by  centrifuging  the  blood  collected  in  the 
citrate  solution,  removing  the  supernatant  fluid,  adding  normal  salt  solution,  and 
centrifuging  again.     Repeat  this  step  once  more  in  order  to  wash  the  cells  thoroughly 
and  after  the  last  centrifuging  remove  the  supernatant  fluid  and  add  sufficient  salt 
solution  to  make  the  total  volume  1  c.c.  and  mix  thoroughly. 

4.  Prepare  an  emulsion  of  staphylococci  which  is  homogeneous  and  free  of  clumps. 

5.  Mark  two  capillary  tubes  with  a  wax  pencil  about  an  inch  from  the  tip;   fit 
rubber  teats  to  the  other  end. 

6.  With  pipet  No.  1  take  up  a  volume  of  blood-cells;   allow  a  bubble  of  air  to 
enter  and  then  an  equal  volume  of  bacterial  emulsion;   bubble  of  air  and  an  equal 
volume  of  the  fresh  unheated  serum.     Mix  well  by  alternate  expulsion  and  aspiration 
on  a  clean  slide.     Then  draw  the  whole  into  the  stem  of  the  pipet  and  seal  the  tip  in 
a  flame. 

7.  Repeat  with  pipet  No.  2,  using  the  heated  serum. 

8.  Incubate  both  pipets  at  37°  C.  for  half  an  hour. 

9.  Remove  the  pipets  from  the  incubator,  break  off  the  tips,  mix  the  contents, 
and  prepare  smears. 

10.  Fix  the  smears  with  a  saturated  solution  of  bichlorid  of  mercury  for  one 
minute;  wash  in  water  and  stain  with  carbol-thionin  for  two  minutes;  wash  in  water 
and  dry. 

11.  Examine  with  oil-immersion  lens. 

(a)  What  are  the  requisites  of  a  satisfactory  opsonic  preparation? 

(b)  Is  there  any  difference  in  the  amount  of  phagocytosis  between 
the  heated  and  unheated  serums?     If  so,  how  do  you  explain  the  result? 

(c)  Do  normal  opsonins  play  any  role  in  natural  immunity? 

(d)  Give  the  properties  of  normal  opsonins. 


908  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

EXPERIMENT  36. — IMMUNE  OPSONINS  (BACTERIOTROPIN) 

1.  Secure  1  c.c.  of  serum  from  a  rabbit  immunized  with  staphylococci  and  heat 
0.5  c.c.  at  56°  C.  for  thirty  minutes. 

2.  Using  the  same  blood  and  bacterial  emulsion  as  prepared  in  the  preceding 
experiment,  prepare  two  opsonic  preparations  with  the  heated  and  unheated  immune 
serum. 

(a)  Is  phagocytosis  more  marked  than  in  the  preceding  experiment? 
If  so,  why? 

(b)  Is  there  any  difference  in  the  degree  and  extent  of  phagocytosis 
with  the  heated  and  unheated  serum? 

(c)  Give  the  properties  of  immune  opsonin  or  bacteriotropin. 

EXPERIMENT  37. — HEMOPSONIN 

1.  Secure  0.5  c.c.  serum  from  a  rabbit  immunized  with  sheep  cells  and  heat  at 
56°  C.  for  half  an  hour  to  destroy  hemolytic  complement. 

2.  Prepare  a  5  per  cent,  suspension  of  sheep  erythrocytes  in  normal  salt  solution 
after  washing  them  three  times. 

3.  Prepare  an  emulsion  of  rabbit  leukocytes,  or  the  emulsion  of  human  cells 
in  the  preceding  experiments  may  be  used. 

4.  Take  a  capillary  pipet;  make  a  mark  about  an  inch  from  the  tip  and  fit  the 
other  end  with  a  rubber  teat. 

5.  Draw  up  equal  volumes  of  leukocytic  emulsion,  sheep  cell  suspension,  and 
serum.     Mix  well,  seal  the  pipet,  and  inoculate  for  an  hour  at  37°  C. 

6.  Prepare  smears  and  stain  with  Wright's  blood-stain. 

(a)  Has  phagocytosis  occurred? 

(b)  Which  cells  have  become  phagocytes? 

(c)  Has  hemolysis  occurred  in  the  mixtures  and  if  not,  why  not? 


EXERCISE  13.— OPSONINS  (Continued) 

EXPERIMENT  38. — MECHANISM  OF  ACTION  OF  OPSONINS 

1.  Secure  serum  from  a  rabbit  immunized  with  Staphylococcus  pyogenes  aureus. 

2.  Prepare  a  leukocytic  suspension  from  normal  rabbit  blood. 

3.  Prepare  an  emulsion  of  eighteen-hour  culture  of  Staphylococcus  aureus. 

4.  Make  the  following  mixtures  in  capillary  pipets: 

No.  1 :  Equal  parts  of  leukocytes,  serum,  and  bacterial  emulsion. 
No.  2:  Equal  parts  of  leukocytes  and  bacterial  suspension. 

5.  In  two  small  test-tubes  mix: 

No.  3:  0.5  c.c.  each  of  leukocytic  suspension  and  serum. 
No.  4:  0.5  c.c.  each  of  serum  and  bacterial  emulsion. 

6.  Incubate  pipets  1  and  2  and  tubes  3  and  4  for  fifteen  minutes  at  37°  C. 

7.  Prepare  smears  of  capillary  tubes  1  and  2  and  stain  with  carbol-thionin. 

8.  To  tubes  3  and  4  add  15  c.c.  normal  salt  solution,  mix,  and  centrifuge  thor- 
oughly.    Decant  off  the  supernatant  fluid  and  restore  volume  in  each  tube  to  1  c.c. 

9.  Prepare  capillary  pipets  as  follows: 


OPSONINS  909 

No.  3:  Equal  parts  of  contents  of  tube  3  and  bacterial  emulsion. 
No.  4:  Equal  parts  of  contents  of  tube  4  and  leukocytic  mixture. 
10.  Incubate  both  pipets  for  fifteen  minutes,  prepare  smears,  and  stain  with  car- 
bol-thionin. 

(a)  Carefully  compare  the  degree  and  extent  of  phagocytosis  in  the 
four  mixtures. 

(b)  Has  leukocytosis  occurred  in  mixture  No.  2  ?     If  so,  what  special 
term  is  applied  to  phagocytosis  in  the  absence  of  serum? 

(c)  What  role   does  serum  play  in    phagocytosis?     Explain    the 
mechanism  involved. 

(d)  Discuss  the  question  of  "stimulin"  and  opsonin  as  demonstrated 
by  the  results  in  preparations  Nos.  3  and  4. 

EXPERIMENT  39. — SPECIFICITY  OF  OPSONINS 

1.  Secure  1  c.c.  of  the  serum  of  a  rabbit  immunized  with  staphylococci  and  heat 
at  56°  C.  for  thirty  minutes.     Divide  into  two  portions  in  separate  small  test-tubes. 

2.  To  No.  1  add  1  c.c.  of  normal  salt  solution  and  4  or  5  loopfuls  of  a  twenty- 
four-hour  agar  slant  culture  of  staphylococci.     Incubate  for  thirty  minutes;  centri- 
fuge thoroughly  and  remove  the  supernatant  fluid  (diluted  serum)  to  a  separate  tube. 
Call  this  "treated"  serum. 

3.  To  the  untreated  serum  add  1  c.c.  salt  solution,  so  that  both  are  diluted 
equally. 

4.  Prepare  an  emulsion  of  rabbit  or  human  leukocytes. 

5.  Prepare  an  emulsion  of  staphylococci,  homogeneous  and  free  of  clumps. 

6.  Prepare  two  opsonic  mixtures:  No.  1  containing  equal  parts  of  blood  suspen- 
sion, bacterial  emulsion,  and  untreated  serum;  No.  2  containing  equal  parts  of  blood, 
bacterial  emulsion,  and  treated  serum. 

7.  Incubate  for  thirty  minutes.     During  this  interval  proceed  as  follows: 

8.  Prepare  an  emulsion  of  typhoid  bacilli,  homogeneous  and  free  of  clumps. 

9.  Secure  0.5  c.c.  of  serum  from  a  rabbit  immunized  with  typhoid  bacilli  and 
heat  at  36°  C.  for  thirty  minutes. 

10.  Prepare  the  following  mixtures  in  capillary  pipets: 

No.  3  blood-cells + typhoid  serum +typhoid  bacterial  emulsion. 

No.  4  blood-cells + typhoid  serum +staphylococcus  bacterial  emulsion. 

No.  5  blood-cells  -{-staphylococcus  serum -f  typhoid  emulsion. 

11.  Incubate  for  fifteen  minutes. 

12.  Prepare  smears  of  all  and  stain  with  carbol-thionin. 

(a)  Is  there  any  difference  in  the  degree  of  phagocytosis  in  mixtures 
No.  1  and  2?     If  so,  why? 

(b)  Are  opsonins  specific?  4 

(c)  Has  phagocytosis  occurred  in  the  cross-mixtures  of  staphylococci 
with  typhoid  serum,  and  typhoid  bacilli  with  staphylococcus  serum?     If 
so,  how  do  you  explain  this  apparent  lack  of  specificity? 

(d)  What  may  happen  in  a  mixture  of  fresh  unheated  typhoid  serum, 
typhoid  bacilli,  and  leukocytic  emulsion? 


910  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

EXERCISE  14.— OPSONIC  INDEX 
EXPERIMENT  40. — DETERMINING  THE  OPSONIC  INDEX 

1.  Secure  a  small  quantity  of  blood  from  a  guinea-pig  or  rabbit  which  has  been 
immunized  with  Staphylococcus  pyogenes  aureus.     Also  two  specimens  from  normal 
animals  to  serve  as  control  serums.     Remove  the  serums,  being  careful  to  keep  them 
marked  and  separate.     The  two  normal  serums  may  be  mixed  in  a  watch-glass  or 
small  test-tube  (pooled). 

2.  Prepare  a  leukocyte  mixture,  using  normal  guinea-pig  or  rabbit  blood  according 
to  the  immune  animal  used. 

3.  Prepare  a  bacterial  emulsion  of  an  eighteen-hour  culture  of  Staphylococcus 
pyogenes  aureus. 

4.  Proceed  to  determine  the  phagocytic  and  opsonic  index  as  per  technic  given  in 
the  text  (page  198). 

(a)  What  constitutes  a  satisfactory  bacterial  emulsion? 

(b)  What  constitutes  a  satisfactory  leukocytic  suspension?     Name 
several  methods  of  obtaining  leukocytes  for  this  technic. 

(c)  What  constitutes  a  satisfactory  phagocytic  film? 

(d)  Should  the  serum  be  fresh? 

(e)  How  do  you  determine  the  phagocytic  index? 

(f)  How  do  you  determine  the  opsonic  index? 

(g)  What  is  the  relation  between  the  opsonic  and  phagocytic  indices? 
(h)  Give  the  practical  value  of  the  opsonic  index  in  disease. 

(i)  Give  the  practical  value  of  the  opsonic  index  as  a  measure  of 
immunity. 

Note. — If  any  of.  the  students  have  received  typhoid  vaccine,  the 
opsonic  index  with  his  serum  may  be  determined. 

EXPERIMENT  41. — QUANTITATIVE  ESTIMATION  OF  BACTERIOTROPINS 

1.  Secure  a  culture  of  pneumococcus. 

2.  Secure  a  few  cubic  centimeters  of  antipneumococcus  serum,  especially  a  serum 
corresponding  to  the  culture. 

3.  Prepare  a  leukocytic  emulsion. 

4.  Conduct  the  test  after  the  technic  given  in  the  text  (page  203). 

(a)  Give  the  value  of  a  bacteriotropic  estimation  of  a  serum. 

(b)  What  relation  do  bacteriotropins  bear  to  immunity? 

(c)  In  what  class  of  diseases  are  bacteriotropins  of  most  value? 

EXERCISE  J5.— BACTERIAL  VACCINES 

EXPERIMENT  42. — PREPARATION  OF  TYPHOID  VACCINE 

1.  Prepare  two  to  six  agar  slant  cultures  of  Bacillus  typhosus  and  grow  for  forty- 
eight  hours  at  37°  C. 

2.  Remove  cultures,  prepare  a  suspension  count  by  the   method   of  Wright, 
sterilize,  dilute,  and  prepare  vaccine  for  administration  as  given  in  the  text. 


ANTITOXINS  911 

3.  Prepare  two  emulsions:  one  to  contain  about  500  million  bacilli  in  each  cubic 
centimeter  (first  dose),  and  the  second  1000  million  per  cubic  centimeter  (second  and 
third  doses). 

EXPERIMENT  43. — PREPARATION  OF  STAPHYLOCOCCUS  VACCINE 

1.  Prepare  two  to  six  agar  slant  cultures  of  Staphylococcus  aureus  and  grow  for 
twenty-four  hours  at  37°  C.     If  a  patient  with  furunculosis  is  available,  make  cultures 
of  pus  and  secure  Staphylococcus,  of  which  a  vaccine  is  prepared. 

2.  Proceed  in  the  preparation  of  the  vaccine  as  given  in  the  text,  placing  each 
dose  in  separate  ampules,  and  so  diluting  that  each  dose  is  of  one  cubic  centimeter 
and  contains  1000  million  of  cocci.     Count  by  the  method  of  Wright  and  by  the 
counting  chamber  method. 


EXERCISE  16.— ANTITOXINS 
EXPERIMENT  44. — STANDARDIZING  DIPHTHERIA  ANTITOXIN 

1.  Prepare  a  strong  diphtheria  toxin  with  the  Park- Williams  Bacillus  No.  8,  after 
the  technic  given  in  the  text. 

2.  Secure  some  of  the  dried  Standard  Antitoxin  and  dilute  so  that  1  c.c.  is  equal 
to  one  immunity  unit. 

3.  With  this  antitoxin  determine  the  L+  dose  of  the  toxin  prepared  according  to 
the  technic  given,  using  six  guinea-pigs  and  the  following  doses  of  toxin:   0.1  c.c., 
0.12  c.c.,  0.15  c.c.,  0.18  c.c.,  0.2  c.c.,  0.25  c.c. 

4.  Secure  a  sample  of  diphtheria  antitoxin  in  the  open  market  containing  about 
4  c.c.  serum  and  2000  units  of  antitoxin.     If  the  titration  given  on  the  label  is  still 
correct,  one  would  expect  about  500  units  of  antitoxin  per  cubic  centimeter  of  serum. 
Carefully  remove  1  c.c.  of  serum  and  dilute  with  19  c.c.  salt  solution  (1:20).     From 
this  stock  dilution  prepare  the  following  dilutions  (taken  from  Bulletin  No.  21, 
Hygienic  Laboratory,  M.  J.  Rosenau): 

1  c.c. -f!4  c.c.  NaCl  solution  1  c.c.  =  .00333  or  ^  =  300  units  per  c.c. 

1  c.c. +16  c.c.  NaCl  solution  1  c.c.  =  .00294  or  ^¥  =  340  units  per  c.c. 

1  c.c. +18  c.c.  NaCl  solution  1  c.c.  =  .00263  or  ^  =  380  units  per  c.c. 

1  c.c.  +20  c.c.  NaCl  solution  1  c.c.  =  .00238  or  ^  =  420  units  per  c.c. 

1  c.c.  +22  c.c.  NaCl  solution  1  c.c.  =  .00217  or  ^  =  460  units  per  c.c. 

1  c.c.  +24  c.c.  NaCl  solution  1  c.&.  =  .002      or  -gfa  =  500  units  per  c.c. 

5.  Mix  1  c.c.  of  these  various  dilutions  with  the  L+dose  of  toxin;  stand  aside 
for  an  hour  and  inject  subcutaneously  in  median  abdominal  line  of  250-  to  300-gram 
guinea-pig  as  per  the  technic  already  given. 

6.  Carefully  observe  all  animals  for  a  period  of  four  days  at  least.     Autopsy 
those  that  succumb,  paying  particular  attention  to  the  condition  of  the  abdominal 
wall  and  suprarenal  glands.     If  the  serum  should  contain  less  than  300  units  of  anti- 
toxin per  cubic  centimeter  of  serum,  the  test  should  be  repeated  with  lower  dilutions. 

7.  Inject  1  c.c.  of  the  serum  subcutaneously  into  a  white  mouse  to  test  for  excess 
of  preservative.     It  requires  1  c.c.  of  a  0.5  per  cent,  solution  of  tricresol  or  0.5  c.c.  of 
a  0.5  per  cent,  phenol  solution  to  kill  a  medium-sized  mouse.     If  the  mouse  shows 
trembling,  it  would  indicate  that  the  serum  contains  nearly  this  percentage  of  tricresol 
(Bulletin  No.  21,  Hygienic  Laboratory). 

8.  Inoculate  1  c.c.  of  the  serum  in  a  flask  containing  100  c.c.  sterile  neutral 
bouillon.     Incubate  at  37°  C.  for-at  least  four  days.     This  will  test  the  sterility  of  the 
product. 


912  EXPERIMENTAL   INFECTION   AND   IMMUNITY 

(a)  Define  the  unit  of  diphtheria  antitoxin. 

(b)  Of  what  practical  value  is  the  measurement  of  diphtheria  anti- 
toxin? 

(c)  Is  there  any  practical  method  of  determining  the  quantity  of 
toxin  in  the  blood  of  a  diphtheric  patient? 

(d)  How  would  you  determine  the  amount  of  natural  antitoxin  in 
the  blood  of  a  person? 

EXPERIMENT  45. — STANDARDIZING  TETANUS  ANTITOXIN 

The  technic  of  standardizing  tetanus  antitoxin  may  be  carried  out  in  a  similar 
manner  with  dried  Standard  Toxin  and  an  antitoxin  purchased  in  the  open  market. 


EXERCISE  \  7.— ANTITOXINS  (Continued) 
EXPERIMENT  46. — SPECIFICITY  OF  ANTITOXINS 

1.  Secure  small  quantities  of  fresh  diphtheria  and  tetanus  toxins;  the  L+dose 
of  each  should  be  known. 

2.  Secure  small  quantities  of  diphtheria  and  tetanus  antitoxins;   the  number  of 
units  per  cubic  centimeter  of  serum  should  be  known. 

3.  Into  four  precision  syringes  place  the  following  mixtures.     After  standing  an 
hour  at  room  temperature,  inject  subcutaneously  into  300-gram  guinea-pigs. 

No.  1:  L+dose  of  diphtheria  toxin +100  units  of  diphtheria  antitoxin.  Inject 
into  pig  No.  1. 

No.  2:  L+  dose  of  diphtheria  toxin +100  units  of  tetanus  antitoxin.  Inject  into 
pig  No.  2. 

No.  3:  L+dose  of  tetanus  toxin +100  units  of  tetanus  antitoxin.  Inject  into 
pig  No.  3. 

No.  4.:  L+  dose  of  tetanus  toxin+100  units  of  diphtheria  antitoxin.  Inject  into 
pig  No.  4. 

(a)  What  do  you  observe  regarding  the  specificity  of  antitoxins? 

(b)  What  are  the  main  symptoms  of  diphtheria  and  tetanus  in  the 
guinea-pig? 

(c)  Even  though  a  pig  injected  with  diphtheria  toxin  shows  no  gen- 
eral symptoms  of  intoxication,  what  local  sign  may  be  present? 

(d)  What  is  the  nature  of  the  toxin-antitoxin  reaction? 

EXPERIMENT    47. — NATURE    OF    THE    TOXIN-ANTITOXIN    REACTION. 
ACTION  OF  ANTI-TETANOLYSIN 

1.  Secure  fresh  tetanus  toxin  and  determine  the  dose  producing  complete  hemoly- 
sis  of  1  c.c.  of  a  1  per  cent,  suspension  of  rabbit  corpuscles  in  two  hours  at  37°  C. 

2.  Place  double  this  dose  of  toxin  in  a  series  of  six  small  test-tubes  and  add  in- 
creasing doses  of  fresh  tetanus  antitoxin:   0.001  c.c.,  0.005  c.c.,  0.01  c.c.,  0.05  c.c., 
0.1  c.c.,  0.2  c.c.   Add  salt  solution  to  bring  the  total  volume  to  1  c.c. ;  incubate  at  37°  C. 
for  an  hour.     Add  1  c.c.  of  1  per  cent,  suspension  of  rabbit  corpuscles  to  each  tube. 
Prepare  two  controls,  one  with  the  dose  of  toxin  and  corpuscles  but  to  which  no  serum 


ANTITOXINS  913 

is  added,  the  second  with  0.2  c.c.  serum  and  dose  of  corpuscles.  Shake  tubes  and 
incubate  for  two  hours.  Make  a  preliminary  reading  and  again  after  tubes  have 
settled  twenty-four  hours  in  the  refrigerator. 

3.  The  first  control  should  be  completely  hemolyzed,  indicating  that  a  sufficient 
lytic  dose  of  toxin  was  employed. 

(a)  What  constituent  of  tetanus  toxin  has  a  marked  affinity  for 
erythrocytes? 

(b)  Is  this  agent  thermostabile?     How  can  you  determine  this? 

(c)  What  role  does  it  play  in  tetanus  intoxication? 

(d)  How  is  this  hemotoxic  agent  neutralized  by  tetanus  antitoxin? 

(e)  Would   anti-tetanospasmin  neutralize  the   hemotoxic   activity 
of  tetanus  toxin? 

(f)  Would  diphtheria  antitoxin  neutralize  this  hemotoxic  agent? 
If  not,  why  not? 

(g)  Explain  the  mechanism  of  neutralization  of  a  toxin  by  antitoxin 
in  vitro?     Is  it  the  same  as  that  occurring  in  vivo? 

EXPERIMENT  48. — ANTISTAPHYLOLYSIN 

1.  Prepare  a  staphylolysin  by  growing  a  culture  of  Staphylococcus  aureus  in 
bouillon  for  two  or  three  weeks.     Pass  through  a  Berkefeld  filter  and  preserve  the 
filtrate  with  0.5  phenol.     Determine  the  lytic  dose  for  1  c.c.  of  a  1  per  cent,  sus- 
pension of  rabbit  corpuscles. 

2.  .Secure  serum  from  a  rabbit  immunized  with  staphylococci.     Heat  at  56°  C. 
for  thirty  minutes. 

3.  Secure  normal  horse  serum.     Heat  it  at  56°  C.  for  thirty  minutes. 

4.  Secure  normal  rabbit  serum.     Heat  at  56°  C.  for  thirty  minutes. 

5.  Into  a  series  of  six  small  test-tubes  place  the  lytic  dose  of  staphylolysin  and 
increasing  amounts  of  rabbit  immune  serum  as  follows:  0.001,  0.005,  0.01,  0.05,  0.1, 
0.2  c.c.     Arrange  a  similar  series,  using  normal  horse  serum  and  normal  rabbit  serum. 
Prepare  a  control  containing  the  lytic  dose  of  toxin.     Add  1  c.c.  of  a  1  per  cent,  sus- 
pension of  rabbit  cells  to  each  tube  and  sufficient  normal  salt  solution  to  make  the 
total  volume  equal  2  c.c.     Shake  each  tube  gently  and  incubate  at  37°  C.  for  two 
hours. 

6.  Inspect  the  tubes.     The  smallest  amount  of  normal  horse  serum  inhibiting 
hemolysis  is  taken  as  1,  or  the  unit.     Compare  the  values  of  the  immune  and  normal 
rabbit  serums  with  this  unit. 

(a)  Why  is  normal  horse  serum  adopted  as  the  standard? 

(b)  What  is  antistaphylolysin? 

(c)  What  are  the  various  agents  produced  by  staphylococci  and  re- 
sponsible for  the  lesions  and  symptoms  of  Staphylococcus  infections? 

(d)  Would  the  antilysin  neutralize  the  leukocidin? 

(e)  Explain  the  mechanism  of  lysin-antilysin  action. 

(f)  Which  role  does  the  lysin  play  in  Staphylococcus  infections? 

(g)  Of  what  value  would  be  the  titration  of  antistaphylolysin  in  the 

serum  of  the  patient? 

58 


914  EXPERIMENTAL   INFECTION   AND    IMMUNITY 


EXERCISE  J8.— FERMENTS  AND  ANTIFERMENTS 
EXPERIMENT  49. — TRYPTIC  FERMENT  OF  LEUKOCYTES 

1.  Collect  0.5  c.c.  of  blood  in  each  of  two  small  test-tubes  by  puncture  of  a  finger; 
set  aside  until  coagulation  has  occurred.     Add  several  changes  of  warm  distilled  water 
until  the  red  corpuscles  are  hemolyzed  and  colorless  clots  of  leukocytes,  fibrin  plate- 
lets, and  detritus  are  secured. 

2.  Place  one  clot  in  the  bottom  of  a  tube  of  sterile  and  slanted  Loeffler  blood- 
serum  medium  which  is  fairly  dry  and  firm. 

3.  Place  the  second  clot  in  a  second  tube  of  this  medium  and  add  0.2  to  0.4  c.c.  of 
fresh  serum. 

4.  Plug  these  tubes  firmly,  paraffin  the  stoppers  to  prevent  evaporation,  and 
incubate  along  with  a  non-inoculated  control  at  50°  C.  for  two  days. 

(a)  In  which  tube  do  you  note  evidences  of  digestion? 

(b)  Describe  the  appearance  of  the  digested  medium. 

(c)  Does  the  tube  containing  serum  show  digestion?     How  do  you 
explain  the  result? 

(d)  What  role  may  this  ferment  play  in  infection? 

(e)  Why  do  not  the  leukocytes  digest  themselves? 

(f)  What  is  the  nature  of  the  ferment? 

(g)  May  this  ferment  play  a  role  in  the  disposal  of  old  and  dead 
leukocytes? 

EXPERIMENT  50. — TESTING  THE  ANTITRYPTIC  POWER  OF  BLOOD- 
SERUM  (AFTER  THE  MARCUS  MODIFICATION  OF  THE  METHOD  OF 
MtJLLER  AND  JOCHMANN) 

1.  Prepare  a  solution  of  trypsin  by  thoroughly  shaking  0.1  gm.  of  Kahlbaum's 
trypsin  with  5  c.c.  glycerin  and  5  c.c.  distilled  water.     Incubate  at  55°  C.  for  half  an 
hour,  shake  thoroughly,  filter,  and  preserve  in  the  refrigerator. 

2.  Secure  six  Petri  dishes  of  Loeffler  blood-serum  culture-media  which  have  been 
sufficiently  dried  to  drive  off  the  water  of  condensation  and  with  a  firm  elastic  surface. 

3.  Collect  1  c.c.  of  blood  from  a  patient  three  hours  after  last  meal;  separate  the 
serum. 

4.  In  a  watch-glass  or  hanging-drop  slide  mix  one  drop  of  serum  with  an  equal 
sized  drop  of  trypsin  solution.     Mix  well  and  transfer  five  loopfuls  to  an  area  on  a 
Petri  dish  of  medium.     With  a  blue-wax  pencil  draw  a  circle  on  the  cover  of  the  plate 
to  include  the  site  of  planting  and  mark  No.  1. 

5.  Prepare  serial  dilutions  with  one  drop  of  serum  with  two,  three,  four,  five,  six, 
seven,  eight,  nine,  and  ten  drops  of  trypsin  solution.     Mix  well  and  plant  five  loop- 
fuls of  each  dilution  on  the  medium  in  five  dishes.     Two  plantings  may  be  made  in  each 
plate  and  with  care  they  will  not  become  confluent.     Mark  each  plate. 

6.  Inoculate  the  sixth  plate  with  five  drops  of  trypsin  solution  without  serum 
(control). 

7.  Incubate  all  tubes  at  37°  C.  for  six  to  twelve  hours  until  the  control  shows 
well-marked  digestion. 

8.  Wherever  the  trypsin  has  not  been  neutralized  by  the  serum,  shallow  liquefied 


FERMENTS  915 

dimples  appear  on  the  surface  of  the  Loeffler  medium.  The  greater  the  amount  of 
trypsin  required  to  cause  digestion,  the  higher  the  titer  of  antitrypsin  in  the  blood. 
By  conducting  a  control  series  with  the  two  normal  pooled  serums  one  may  determine 
whether  in  a  given  case  the  antitryptic  power  of  the  blood  is  normal,  increased,  or 
decreased. 

(a)  Does  this  test  possess  any  practical  value? 

(b)  Discuss  the  presence  of  antitrypsin  in  the  blood-serum. 


EXERCISE  19.— FERMENTS  (Continued) 

EXPERIMENT  51. — ABDERHALDEN  SEROENZYME  REACTION  IN  PREG- 
NANCY 

The  technic  of  this  test  is  very  exact.  The  glassware  should  be 
sterile  and  all  manipulations  carried  out  carefully  and  exactly  as  laid 
down  by  Abderhalden.  Consult  the  text  for  the  technic  (Chapter 

XV). 

1.  Test  five  Schleichter  and  Schull  shells  No.  579a  for  permeability  to  albumin, 
using  5  per  cent,  egg-albumen  water  and  the  biuret  or  ninhydrin  reaction. 

2.  Those  shells  which  prove  impermeable  to  albumin  are  now  tested  with  a  1 
per  cent,  solution  of  silk  peptone  (Hochst),  using  ninhydrin  as  the  indicator.     The 
shells  impermeable  to  albumin  and  permeable  to  peptone  are  suitable  for  the  main 
test. 

3.  Secure  a  fresh  human  placenta;  wash  thoroughly  to  remove  blood;   cut  into 
pieces  about  the  size  of  a  dime;  wash  and  rewash  until  perfectly  white;  search  care- 
fully for  tiny  blood-clots;   boil  repeatedly  until  the  water  reacts  negatively  to  nin- 
hydrin.    Consult  the  text-book  for  the  exact  technic.     Abderhalden  lays  a  great 
deal  of  stress  upon  the  proper  preparation  of  the  substratum. 

4.  Secure  blood  from  a  patient  in  advanced  pregnancy;   also  a  specimen  from  a 
male.     After  a  few  hours,  separate  the  serums  and  centrifuge  thoroughly  until  free 
of  cells.     The  serums  should  not  be  over  twelve  hours  old. 

5.  Conduct  a  reaction  after  the  technic  given  in  the  text-book,  including  the 
controls. 

(a)  Describe  the  biuret  and  ninhydrin  reactions. 

(b)  Why  must  the  shells  be  so  carefully  tested? 

(c)  Why  must  the  placenta  be  free  of  blood  and  boiled  previous  to 
the  test? 

(d)  Why  should  an  aseptic  technic  be  employed? 

(e)  What  are  the  prevailing  views  regarding  the  specificity  of  the 
ferments? 

(f)  Has  the  pregnancy  test  a  practical  value? 

(g)  Give  the  principles  of  the  optical  method, 
(h)  Why  is  the  serum  control  so  important? 

(i)  Enumerate  the  principal  sources  of  error  in  the  technic  of  the 
dialysation  method. 


916  EXPERIMENTAL   INFECTION    AND    IMMUNITY 

EXERCISE  20.— AGGLUTININS 

EXPERIMENT  52. — THE  GRUBER-WIDAL  REACTION  IN  TYPHOID  FEVER 
("WET"  METHOD).     MICROSCOPIC  REACTION 

1.  Collect  blood  in  a  Wright  capsule  or  small  test-tube  from  a  typhoid  conva- 
lescent patient.     Rabbit  immune  serum  may  be  used  instead.     Separate  the  serum 
from  the  clot. 

2.  Prepare  two  dilutions  with  hanging-drop  slides,  1 : 50  and  1 : 100,  and  a  culture 
control.     Use  a  twenty-four-hour  bouillon  culture  of  Bacillus  typhosus — one  free  of 
clumps  and  in  which  the  bacilli  are  long  and  motile. 

3.  It  is  well  to  prepare  a  similar  test  with  a  known  positive  typhoid  serum  and 
a  normal  negative  serum. 

4.  Dilution  and  slides  are  prepared  according  to  the  technic  given  in  the  text- 
book.    A  second  set  of  tests  in  dilutions  of  1 : 40  and  1 : 80  may  be  prepared  with  the 
aid  of  the  white  corpuscle  pipet. 

5.  Place  slides  away  from  direct  sunlight  and  examine  at  the  end  of  an  hour. 
Examinations  are  readily  made  with  the  y§  objective,  the  light  being  well  cut  off. 
Examine  the  culture  control  first,  then  the  higher  and  lower  dilutions. 

(a)  Describe  the  phenomenon  of  agglutination. 

(b)  Is  it  necessary  for  all  bacilli  to  be  agglutinated  to  constitute  a 
positive  reaction? 

(c)  What  are  the  features  of  a  doubtful  reaction?     Of  a  negative  re- 
action? 

(d)  Does  the  normal  serum  contain  agglutinin  for  the  typhoid  ba- 
cillus? 

(e)  Why  is  it  necessary  to  use  high  dilutions?     What  dilution  is  the 
lower  limit  of  practical  safety? 

(f)  Does  the  control  show  agglutination?     If  so,  by  what  term  is 
this  agglutination  known? 

(g)  What  are  the  characteristics  of  a  satisfactory  culture  for  the 
microscopic  agglutination  test? 

(h)  Would  a  dead  culture  be  serviceable  in  this  reaction? 

(i)  What  practical  value  has  the  Widal  reaction  in  the  diagnosis  of 
typhoid  fever? 

(j)  What  value  has  the  agglutination  reaction  in  determining  the 
degree  of  immunity  following  typhoid  immunization? 

Note. — If  the  students  are  immunized  with  typhoid  vaccine,  they 
may  use  one  another's  serum  in  this  and  following  experiments. 

EXPERIMENT  53. — GRUBER-WIDAL    REACTION    IN    TYPHOID    FEVER 

("DRY"  METHOD) 

1.  Prepare  smears  of  blood  of  a  typhoid  convalescent  patient  on  clean  glass 
slides  or  collect  a  few  drops  on  partially  glazed  paper,  as  prescription  blanks.  Allow 
blood  to  dry  and  do  not  apply  heat. 


AGGLUTININS  917 

2.  Using  a  good  twenty-four-hour-old  culture  of  Bacillus  typhosus,  prepare  an 
agglutination  test  after  the  technic  given  hi  the  text-book.     Be  particularly  careful 
not  to  transfer  paper  fiber  to  the  slide — apply  the  salt  solution  and  dissolve  the  blood 
by  gently  rubbing  with  a  small  platinum  loop  (2  mm.).     Mix  the  blood  in  the  loop- 
ful  of  culture  with  the  slide  held  or  placed  over  a  white  surface  so  that  the  proper 
delicate  orange  tint  is  secured. 

3.  Prepare  culture  control  as  usual;  also  similar  tests  with  a  known  positive  and 
negative  serum. 

4.  Examine  at  the  end  of  an  hour. 

(a)'  What  special  precautions  are  to  be  observed  in  this  method? 

(b)  What  is  the  particular  practical  value  of  this  reaction? 

(c)  Are  accurate  dilutions  possible  and  if  so,  by  what  technic? 

(d)  Are  agglutinins  resistant  to  deleterious  influences? 

(e)  Compare  the  value  of  this  method  with  the  one  used  in  the  pre- 
ceding experiment. 

EXERCISE  2 J.— AGGLUTININS  (Continued) 
EXPERIMENT  54. — MACROSCOPIC  AGGLUTINATION  REACTION 

1.  Secure  serum  from  a  rabbit  which  has  been  immunized  with  Bacillus  typhosus. 
Serum  of  a  typhoid  convalescent  may  be  used  instead. 

2.  Prepare  a  series  of  serum  dilutions  in  small  narrow  test  tubes  in  amounts  of 
1  c.c.,  ranging  from  1: 10  up  to  1:640. 

3.  Add  to  each  1  c  c.  of  the  bacillary  emulsion  of  a  good  twenty-four-  to  forty- 
eight-hour  bouillon  culture  of  Bacillus  typhosus  or  an  emulsion  prepared  by  washing 
twenty-four-hour  growths  from  agar  slant  cultures  with  normal  salt  solution,  accord- 
ing to  the  technic  given  in  the  text.     This  doubles  the  dilutions,  which  now  range 
from  1 :  20  up  to  1 : 1280.     Prepare  the  culture  control. 

4.  Shake  gently  and  incubate  for  two  hours  at  37°  C.  and  then  record  results  after 
tubes  have  been  standing  at  room  temperature  for  six  hours.     Reexamine  tubes  with 
the  agglutinoscope  and  note  the  higher  delicacy  of  such  readings. 

5.  If  a  typhoid  immune  serum  of  unknown  titer  is  used  and  agglutination  is 
complete  in  the  highest  dilution,  the  test  must  be  repeated  with  still  higher  dilutions 
in  order  to  determine  the  agglutinin  titer  of  the  serum. 

(a)  Has  agglutination  occurred  in  the  control  tube?     Why  is  this 
control  so  important  in  this  and  all  agglutination  reactions? 

(b)  Is  agglutination  as  complete  in  the  lower  as  in  the  higher  reac- 
tion?    If  not,  how  do  you  explain  this  result?     Of  what  practical  im- 
port is  this  phenomenon? 

(c)  Describe  the  appearance  of  macroscopic  agglutination. 

(d)  Are  the  agglutinated  bacilli  dead?     To  determine  this,  pipet  off 
the  supernatant  fluids  of  several  tubes  into  a  germicidal  solution;   then 
add  an  excess  of  sterile  normal  salt  solution  to  the  sediment  of  aggluti- 
nated bacilli,  stir  up  the  sediment,  and  transfer  with  a  sterile  pipet  to  a 
sterile  centrifuge  tube;   centrifuge  thoroughly;   remove  the  supernatant 


918  EXPERIMENTAL  INFECTION   AND    IMMUNITY 

fluid  with  a  sterile  pipet  and  plant  several  loopf  uls  of  bacteria  on  slants  of 
agar  and  in  neutral  bouillon.     Why  is  it  advisable  to  wash  the  sediment? 
(e)  What  advantages  has  the  macroscopic  over  the  microscopic 
method? 

EXPERIMENT  55. — MACROSCOPIC  AGGLUTINATION  REACTION   (KOLLE) 

1.  Prepare  dilutions  in  amounts  of  1  c.c.  of  a  typhoid  immune  serum  in  proper 
test-tubes,  ranging  from  1 : 20  up  to  1 : 1280. 

2.  Add  one  loopf ul  of  a  twenty-four-hour  culture  of  Bacillus  typhosus  to  each 
tube,  being  careful  to  emulsify  thoroughly  on  the  side  of  the  test-tube  according  to  the 
technic  given.     This  does  not  materially  alter  the  degree  of  dilution.     Prepare  the 
culture  control  as  usual. 

3.  Incubate  and  examine  tubes  as  in  the  previous  experiment. 

What  are  the  advantages  and  disadvantages  of  this  method? 

EXPERIMENT  56. — MACROSCOPIC  AGGLUTINATION  REACTION   (KILLED 
CULTURE) 

1.  Inoculate  a  flask  containing  200  c.c.  of  neutral  broth  with  Bacillus  typhosus 
and  incubate  for  forty-eight  hours.     At  the  end  of  this  time  a  good  rich  growth  is 
usually  secured.     Shake  gently  to  stir  and  break  up  any  clumps  of  bacilli  and  heat 
at  60°  C.  for  one  hour  on  a  water-bath,  gently  shaking  the  flask  once  or  twice  during 
this  time.     Add  5  c.c.  formalin;  shake,  and  place  in  the  refrigerator  for  three  days; 
stopper  the  flask  with  a  rubber  stopper,  and  before  using  shake  well  in  order  thor- 
oughly to  mix  the  dead  bacilli  which  drop  to  the  bottom  of  the  flask. 

2.  Prepare  a  series  of  dilutions  of   typhoid   immune  serum  and  conduct  this 
experiment  according  to  the  macroscopic  technic,  using  the  dead  instead  of  a  living 
bacillary  emulsion. 

(a)  What  are  the  advantages  of  using  a  killed  culture? 

(b)  Is  this  method  as  delicate  as  when  living  cultures  are  used? 

(c)  Is  spontaneous  agglutination  likely  to  occur? 

(d)  What  constitutes  a  satisfactory  killed  culture  for  this  reaction? 


EXERCISE  22.— AGGLUTININS  (Continued) 
EXPERIMENT  57. — GROUP  AGGLUTINATION 

1.  Prepare  five  series  of  test-tubes  containing  1  c.c.  of  dilutions  ranging  from  1:20 
to  1 : 640  of  a  typhoid  immune  serum. 

2.  To  the  first  series  add  1  c.c.  of  a  thirty-six-hour  bouillon  culture  of  Bacillus 
typhosus;   to  the  second  series,  Bacillus  paratyphosus  "B";  to  the  third,  Bacillus 
paratyphosus  "A";  to  the  fourth,  Bacillus  enteritidis  (Gartner);  to  the  fifth,  Bacillus 
coli.     The  dilutions  are  thus  doubled.     Prepare  culture  controls  of  all  five  cultures, 
and  be  careful  that  tubes  are  properly  labeled. 

3.  Incubate  for  two  hours  and  record  reactions  at  the  end  of  further  six  hours  at 
room  temperature. 

4.  This  experiment  may  be  repeated  with  the  serum  of  a  typhoid  convalescent 
patient. 


AGGLUTININS  919 

(a)  In  which  series  of  tubes  is  agglutination  most  complete? 

(b)  Discuss  the  question  of  specificity  of  the  agglutinins. 

(c)  How  do  you  explain  group  agglutination? 

(d)  Are  group  agglutinins  present  to  the  same  degree  as  the  main 
agglutinin?     How  may  the  group  agglutinins  be  eliminated? 

(e)  Of  what  value  is  the  agglutination  reaction  in  showing  the  bio- 
logic relationship  of  bacteria? 

(f)  How  would  the  agglutination  reaction  be  used  in  the  diagnosis 
of  an  unknown  microorganism? 

EXPERIMENT  58. — PRO-AGGLUTINATION — AGGLUTINOIDS 

1.  This  very  important  phase  of  agglutination  may  have  been  noted  in  the  pre- 
vious experiments,  especially  if  old  immune  serums  were  used,  when  there  is  a  possi- 
bility that  agglutinin  has  degenerated  in  agglutinoids.     Agglutinoids  having  a  stronger 
affinity  than  agglutinin  for  the  agglutinogen,  and  having  lost  the  agglutinophore 
group,  produce  little  or  no  agglutination  and  prevent  the  action  of  agglutinin  until 
diluted  out  of  action  in  the  higher  serum  dilutions.     In  this  way  agglutination  may  be 
poor  or  absent  in  low  and  present  in  higher  dilutions,  an  important  practical  fact 
to  be  remembered. 

2.  The  action  of  agglutinoids  may  be  seen  by  conducting  a  macroscopic  test  with 
an  old  immune  serum. 

3.  Otherwise  some  agglutinoid  may  be  produced  by  heating  an  immune  serum 
to  60°  C.  for  an  hour  on  the  water-bath  and  conducting  the  test  with  dilutions  of  the 
heated  serum. 

4.  Repeat  the  tests  with  old  and  heated  immune  serums  in  dilutions  of  1:40, 
1:80,  1:160,  1:320,  using  the  macroscopic   and   microscopic  technic,  as  this  phe- 
nomenon of  pro-agglutination  is  not  infrequently  found  in  the  routine  Widal  reaction 
in  typhoid  fever. 

EXPERIMENT  59. — THE  ABSORPTION'  OR  SATURATION  AGGLUTINATION 
REACTION  (CASTELLANI) 

1.  Immunize  a  rabbit  with  three  intravenous  injections  of  a  heated  emulsion  of 
Bacillus  typhosus  and  three  of  Bacillus  paratyphosus  "B"  or  Bacillus  coli,  according 
to  the  technic  given  under  Active  Immunization  and  Methods  of  Animal  Inoculation. 

2.  With  this  immune  serum  conduct  a  test  according  to  the  technic  given  in  the 
text. 

(a)  Of  what  practical  value  is  this  method? 

(b)  What  are  "major"  and  " minor"  agglutinins? 


EXERCISE  23.— AGGLUTININS  (Continued) 
EXPERIMENT  60. — HEMAGGLUTININS 

1.  Secure  0.1  c.c.  of  antihuman  hemolysin  prepared  by  giving  a  rabbit  a  series 
of  injections  of  washed  human  erythrocytes.     Inactivate  the  serum  by  heating  to 
55°  C.  for  half  an  hour.     Dilute  1: 100  by  adding  9.9  c.c.  salt  solution. 

2.  Prepare  10  c.c.  of  a  1  per  cent,  suspension  of  washed  human  erythrocytes. 


920  EXPERIMENTAL  INFECTION  AND    IMMUNITY 

3.  Into  a  series  of  six  small  test-tubes  place  0.1  c.c.,  0.2  c.c.,  0.4  c.c.,  0.6  c.c.,  0.8 
c.c.,  1  c.c.  of  the  diluted  immune  serum;  add  1  c.c.  of  suspension  of  blood-cells  and 
1  c.c.  of  salt  solution  to  each  tube.     As  a  control,  place  1  c.c.  of  corpuscle  suspension 
and  1  c.c.  of  normal  salt  solution  into  a  seventh  tube. 

4.  Shake  gently  and  place  in  the  incubator  for  an  hour. 

(a)  Describe  agglutination  of  red  corpuscles. 

(b)  Are  the  clumps  easily  broken  up? 

(c)  What  would  have  occurred  if  complement  were  present? 

(d)  Why  was  it  necessary  to  heat  the  fresh  immune  serum?     Is  it 
necessary  to  heat  an  old  immune  serum? 

(e)  Examine  a  loopful  of  the  sedimented  corpuscles  microscopically. 

EXPERIMENT  61. — BLOOD  TRANSFUSION  TESTS 

Several  students  may  contribute  a  few  cubic  centimeters  of  their  blood  and 
agglutination  and  hemolysin  tests  made  as  described  in  the  text  (Chapter  XVI). 

Of  what  value  are  agglutination  and  hemolytic  tests -preliminary  to 
blood  transfusion? 


EXERCISE  24.— PRECIPITINS 
EXPERIMENT  62. — TITRATION  OF  A  PRECIPITIN  SERUM 

1.  Secure  1  c.c.  of  antihorse  immune  serum  prepared  by  immunizing  a  rabbit 
with  normal  horse  serum. 

2.  Secure  1  c.c.  of  normal  horse  serum  and  place  2  c.c.  of  the  following  dilutions 
made  with  normal  salt  solution  into  a  series  of  six  narrow  test-tubes :  1 : 100,  1 : 500, 
1:1000,  1:2000,  1:5000,  and  1:10,000. 

3.  To  each  tube  add  0.1  c.c.  of  the  immune  serum.     The  tubes  must  not  be 
shaken. 

4.  Place  tubes  in  an  incubator  at  37°  C.  and  observe  every  thirty  minutes  for 
two  hours  and  again  after  standing  in  a  refrigerator  overnight. 

(a)  Describe  the  phenomenon  of  precipitation. 

(b)  What  are  the  requisites  of  a  satisfactory  precipitin  serum? 

(c)  What  are  the  requisites  of  a  satisfactory  preparation  of  the 
antigen  for  this  reaction? 

(d)  Discuss  the  delicacy  of  this  reaction. 

EXPERIMENT  63. — TITRATION  OF  A  PRECIPITIN  SERUM 

Repeat  this  titration,  using  antihuman  immune  serum  and  normal  human 
serum. 

EXPERIMENT  64. — SPECIFICITY  OF  PRECIPITINS 

1.  Secure  1  c.c.  each  of  clear  antihorse  and  antihuman  serums. 

2.  Secure  1  c.c.  each  of  normal  horse,  normal  human,  and  normal  guinea-pig 


PRECIPITINS  921 

serum.     Prepare  1:100  dilutions  with  normal  salt  solution;  set  up  the  following  in 
long  narrow  test-tubes: 

Tube  1:  2  c.c.  of  normal  horse  serum  (1:100)4-0.1  c.c.  antihorse  serum. 
Tube  2:  2  c.c.  of  normal  horse  serum  (1: 100) +0.1  c.c.  antihuman  serum. 
Tube  3:  2  c.c.  of  normal  human  serum  (1:100) +0.1  c.c.  antihuman  serum. 
Tube  4:  2  c.c.  of  normal  human  serum  (1:100) +0.1  c.c.  antihorse  serum. 
Tube  5:  2  c.c.  of  normal  guinea-pig  serum  (1:100) +0.1  c.c.  antihorse  serum. 
Tube  6:  2  c.c.  of  normal  guinea-pig  serum  (1: 100) +0.1  c.c.  antihuman  serum. 
Tube  7:  2  c.c.  of  normal  salt  solution+0.1  c.c.  antihorse  serum  (control). 
Tube  8:  2  c.c.  of  normal  salt  solution+0.1  c.c.  antihuman  serum  (control). 

3.  Do  not  shake  the  tubes;  place  them  in  the  incubator  at  37°  C.;  inspect  every 
thirty  minutes  for  two  hours. 

(a)  Discuss  the  question  of  specificity  of  precipitins. 

(b)  Of  what  practical  value  are  precipitin  reactions? 

(c)  Enumerate  the  chief  points  in  the  technic  of  a  precipitin  reac- 
tion in  the  differentiation  of  proteins. 


EXERCISE  25.— PRECIPITINS  (Continued) 
EXPERIMENT  65. — FORENSIC  BLOOD  TEST 

1.  Secure  from  the  instructor  two  pieces  of  muslin  or  gauze  containing  respec- 
tively a  stain  of  human  and  horse  blood.   These  are  to  be  numbered  and  the  source  of 
each  stain  known  only  to  the  instructor.     Secure  1  c.c.  each  of  normal  human  and 
horse  serum  and  dilute  1 : 1000. 

2.  Prepare  extracts  of  each  stain  as  described  in  the  text  (Chapter  XVII). 

3.  Secure  1  c.c.  of  clear  antihuman  and  antihorse  immune  serum. 

4.  Set  up  the  following  in  long  narrow  test-tubes: 

Tube  1 :  2  c.c.  of  extract  No.  1  in  dilution  of  1 : 1000+0.1  c.c.  of  antihuman  serum. 

Tube  2:  2  c.c.  of  extract  No.  1  in  dilution  of  1 : 1000+0.1  c.c.  of  antihorse  serum. 

Tube  3:  2  c.c.  of  extract  No.  2  in  dilution  of  1 : 1000+0.1  c.c.  of  antihorse  serum. 

Tube  4:  2  c.c.  of  extract  No.  2  in  dilution  of  1 : 1000+0.1  c.c.  of  antihuman  serum. 

Tube  5:  2  c.c.  of  normal  human  serum  in  dilution  1:1000+0.1  c.c.  antihuman 
serum  (control). 

Tube  6:  2  c.c.  of  normal  horse  serum  in  dilution  of  1:1000+0.1  c.c.  antihorse 
serum  (control). 

Tube  7:  2  c.c.  of  normal  salt  solution+0.1  c.c.  of  antihuman  serum. 

Tube  8:  1  c.c.  of  normal  salt  solution+0.1  c.c.  of  antihorse  serum. 

5.  Do  not  shake  tubes;  place  in  the  incubator  at  37°  C.  and  inspect  every  thirty 
minutes  for  two  hours  and  after  standing  in  the  refrigerator  overnight. 

(a)  Inspect  the  controls.     Are  the  antiserums  potent  and  satis- 
factory?    Why  is  it  advisable  to  have  these  controls? 

(b)  Are  you  able  to  diagnose  the  source  of  each  stain? 

(c)  In  a  medicolegal  test,  if  these  reactions  were  negative,  how  would 
you  proceed  further  in  your  efforts  to  establish  the  identity  of  a  particu- 
lar stain? 


922  EXPERIMENTAL   INFECTION   AND    IMMUNITY 


EXERCISE  26,— PRECIPITINS  (Continued) 
EXPERIMENT  66. — MILK-PRECIPITIN  (LACTOSERUM) 

1.  Secure  a  cubic  centimeter  of  anticow  serum  previously  prepared  by  immuniz- 
ing rabbits  with  injections  of  fresh  cow's  milk. 

2.  Prepare  a  1:50  dilution  of  cow's  milk  (precipitinogen)  by  mixing  0.2  c.c.  milk 
with  9.8  c.c.  normal  salt  solution.     Prepare  a  similar  dilution  of  human  or  goat  milk 
for  control  tests. 

3.  Into  a  series  of  six  small  test-tubes  place  2  c.c.  of  the  following  dilutions  of 
cow's  milk:    1:50,  1:100,  1:200,  1:400,  1:800,  and  1:1600. 

4.  Into  a  second  series  of  three  tubes  place  2  c.c.  of  the  following  dilutions  of 
control  milk :  1 : 50,  1 : 100,  1 : 200. 

5.  Into  the  nine  tubes  of  these  series  add  0.1  c.c.  cow  lactoserum. 

6.  A  third  series  of  six  tubes  containing  2  c.c.  of  the  above  dilutions  of  cow's 
milk  plus  0.1  c.c.  of  normal  rabbit  serum  may  be  set  up  as  further  controls. 

7.  Keep  the  tubes  at  room  temperature  and  inspect  at  the  end  of  fifteen  min- 
utes; half  an  hour;  one  hour,  and  two  hours. 

(a)  Describe  a  positive  reaction. 

(b)  Discuss  the  specificity  of  lactoserums. 

(c)  Of  what  practical  value  are  lactoserum  reactions? 

EXPERIMENT  67. — BACTERIAL  PRECIPITINS 

1.  Inoculate  two  flasks  each  containing  50  c.c.  of  sterile  neutral  bouillon  with 
cultures  of  Bacillus  typhosus  and  Bacillus  coli  and  cultivate  at  37°  C.  for  three  weeks. 
Filter  cultures  through  a  sterile  Berkefeld  filter  until  clear. 

2.  Into  a  series  of  four  small  test-tubes  place  2  c.c.  of  the  following  dilutions  of 
the  typhoid  filtrate  (precipitinogen)  undiluted:    1:2,    1:4,  and  1:10.     Prepare  a 
second  series  of  tubes  with  similar  amounts  of  the  same  dilutions  of  Bacillus  coli  filtrate. 

3.  Add  0.1  c.c.  of  potent  typhoid  immune  serum  to  all  tubes  of  both  series.     A 
serum  with  high  agglutinin  titer  will  be  satisfactory. 

4.  Place  2  c.c.  of  the  undiluted  typhoid  and  coli  filtrates  in  separate  tubes  as 
controls.     Prepare  an  additional  control  by  placing  0.1  c.c.  of  the  typhoid  immune 
serum  in  2  c.c.  normal  salt  solution. 

5.  Observe  the  tubes  at  the  end  of  half  an  hour,  and  after  one,  two,  and  six 
hours. 

(a)  Describe  a  positive  bacterial  precipitin  reaction. 

(b)  Has  a  precipitate  formed  with  the  Bacillus  coli  nitrate?     With 
what  microorganism  would  typhoid  immune  serum  be  likely  to  produce 
a  precipitate? 

(c)  Discuss  the  relative  delicacy  of  precipitation  and  agglutination 
reactions  in  the  differentiation  of  bacteria. 

EXPERIMENT  68. — NOGUCHI  GLOBULIN  REACTION 

1.  Secure  1  c.c.  each  of  several  cerebrospinal  fluids  from  cases  of  paresis,  tubercu- 
lous meningitis,  serous  meningitis,  and  normal  persons. 

2.  Conduct  this  floccule-forming  reaction  after  the  technic  given  in  the  text. 

3.  Make  total  cell  counts  on  each  fluid  (see  text). 


AMBOCEPTORS   AND    COMPLEMENTS. — HEMOLYSINS  923 

(a)  Explain  the  appearance  of  a  positive  reaction. 

(b)  What  constitutes  a  negative  reaction? 

(c)  What  is  the  diagnostic  value  of  this  reaction  in  syphilis? 

(d)  What  other  value  has  this  reaction  in  diagnosis? 

(e)  What  relation  does  this  reaction  bear  to  total  cell  counts? 

(f)  Explain  the  mechanism  of  the  reaction. 


EXERCISE  27.— AMBOCEPTORS  AND  COMPLEMENTS.— HEMOLYSINS 

EXPERIMENT  69. — RESISTANCE  OF   RED  BLOOD-CORPUSCLES  TO  SALT 
SOLUTION  OF  VARIOUS  TONICITIES.  NON-SPECIFIC  HEMOLYSIS 

1.  Arrange  a  series  of  twenty-three  small  sterile  test-tubes  in  a  rack;  to  each  of 
the  first  twenty-one  tubes  add  3  c.c.  of  various  dilutions  of  salt  solutions  ranging 
from  0.6  per  cent,  to  0.2  per  cent,  in  steps  of  0.02  per  cent.     Large  amounts  of  these 
solutions  should  be  at  hand,  preserved  in  bottles  fitted  with  tight  cork  stoppers  to 
prevent  evaporation,  or  prepared  at  the  time  according  to  the  technic  given  in  the 
text  (page  402).     To  tube  No.  22  add  3  c.c.  distilled  water  and  to  No.  23  the  same 
amount  of  normal  salt  solution  (0.85  per  cent.). 

2.  Secure  blood-corpuscles  after  the  method  given  in  the  text.     Dog  blood  may 
be  substituted  for  human  blood.     After  the  final  washing,  add  0.05  c.c.  to  each  tube. 
Shake  gently. 

3.  Make  a  preliminary  reading  at  once;  the  final  reading  is  made  after  the  tubes 
have  been  standing  hi  the  refrigerator  overnight. 

4.  Note  the  appearance  of  hemolyzed  blood.     Prepare  a  color  scale  by  placing 
1  c.c.  blood-corpuscles  in  30  c.c.  distilled  water,  which  represents  a  standard  of  100 
per  cent,  hemolysis.     From  this  solution  prepare  dilutions  with  distilled  water  to 
represent  80,  60,  40  and  20  per  cent,  hemolysis.     For  example,  4  c.c.  of  the  stock 
solution  plus  1  c.c.  distilled  water  equals  5  c.c.  of  an  80  per  cent,  solution;  3  c.c.  of 
the  stock  plus  2  c.c.  water  equals  a  60  per  cent,  solution;  2  c.c.  of  stock  plus  3  c.c.  of 
water  equals  a  40  per  cent,  solution,  and  so  on.     Carefully  compare  the  volume  of 
non-hemolyzed  cells  in  the  bottom  of  some  of  the  test-tubes  with  the  amount  of  color 
in  the  supernatant  fluid.     A  Duboscq  colorimeter  may  be  used  for  making  com- 
parisons with  the  controls. 

5.  Determine  the  minimal  and  maximal  resistance  of  the  corpuscle  employed. 

(a)  What  is  the  appearance  of  hemolyzed  blood? 

(b)  What  is  the  meaning  of  the  term  hemolysis? 

(c)  Why  is  this  called  non-specific  hemolysis? 

(d)  What  does  a  normal  salt  solution  mean? 

(e)  What  is  the  importance  of  using  a  normal  salt  solution  in  hemo- 
lytic  experiments? 

(f)  How  is  hemolysis  produced  by  hypotonic  solutions? 

(g)  What  would  be  the  objections  of  using  a  hypertonic  salt  solution? 
(h)  How  is  an  isotonic  salt  solution  prepared? 

(i)    What  is  the  meaning  of  the  terms  minimal  and  maximal  re- 
sistance of  red  blood-corpuscles? 

(j)    Of  what  practical  value  is  this  test? 


924  EXPERIMENTAL  INFECTION  AND  IMMUNITY 

EXPERIMENT  70. — THE  HEMOLYTIC  INFLUENCE  OF  ACIDS  AND  ALKALIS 
The  object  of  this  experiment  is  to  demonstrate  the  hemolytic 
activity  of  acids  and  alkalies  as  bearing  upon  the  question  of  clean  glass- 
ware in  hemolytic  work,  and  particularly  complement-fixation  tests. 

1.  Into  a  series  of  six  test-tubes  place  increasing  amounts  of  a  solution  of  hydro- 
chloric acid  prepared  by  diluting  1  c.c.  of  the  normal  solution  with  99  c.c.  of  normal 
salt  solution:  0.2,  0.4,  0.6,  0.8,  1.0,  and  2.0  c.c.  respectively. 

2.  Into  a  second  series  of  six  test-tubes  place  increasing  amounts  of  a  solution  of 
sodium  hydroxid  prepared  by  diluting  1  c.c.  of  the  normal  solution  with  99  c.c.  of 
normal  salt  solution:   0.2,  0.4,  0.6,  0.8,  1.0,  and  2.0  c.c.  respectively. 

3.  Make  the  total  volume  in  each  tube  equal  2  c.c.  with  the  addition  of  normal 
salt  solution. 

4.  To  each  tube  and  a  normal  salt  solution  control  add  1  c.c.  of  a  2|  per  cent, 
suspension  of  washed  sheep  cells.    Mix  and  stand  aside  for  an  hour  or  two. 

(a)  Has  hemolysis  occurred  in  any  of  the  tubes? 

(b)  What  are  the  smallest  amounts  of  acid  and  alkali  producing 
complete  hemolysis  in  this  experiment? 

(c)  Of  what  practical  significance  are  these  results? 

(d)  How  should  test-tubes  be  prepared  for  hemolytic  work? 

EXPERIMENT  71. — SERUM  HEMOLYSIS  IN  VITRO 

1.  Secure  2  c.c.  of  blood  from  the  ear  of  a  rabbit  which  has  received  at  least  two 
intravenous   injections   of   sheep  cells.     Separate  the  serum  and  divide  into  two 
portions.     Inactivate  one  portion  (A)  by  heating  in  a  water-bath  for  half  an  hour  at 
56°  C. 

2.  Place  0.1  c.c.  and  0.2  c.c.  of  the  fresh  unheated  serum  of  portion  (B)  in  two 
test-tubes.     Likewise  the  same  amounts  of  the  heated  serum  (A)  in  two  more  tubes. 
Add  1  c.c.  of  a  1  per  cent,  suspensior  of  washed  sheep  cells  to  each  tube  and  sufficient 
salt  solution  to  bring  the  total  volume  to  3  c.c.     As  a  control,  place  1  c.c.  of  corpuscle 
suspension  and  3  c.c.  salt  solution  in  a  tube.     Shake  gently,  and  incubate  for  an 
hour. 

(a)  Which  tubes  show  hemolysis? 

(b)  What  substances  are  concerned  in  serum  hemolysis? 

(c)  Why  did  hemolysis  occur  with  the  unheated  and  not  with  the 
heated  serum?     What  is  the  meaning  of  inactivation  of  a  serum? 

(d)  Why  is  this  called  serum  hemolysis? 

(e)  What  is  the  appearance  of  the  control  tube?     Why  is  this  con- 
trol included?     What  would  have  happened  if  a  hypotonic  salt  solution 
had  been  used? 

EXPERIMENT  72. — SERUM  HEMOLYSIS  IN  Vivo 

1.  Immunize  a  rabbit  with  three  intravenous  injections  of  5  c.c.  each  of  a  10  per 
cent,  suspension  of  washed  cat  erythrocytes  in  sterile  salt  solution.     Give  the  in- 


AMBOCEPTORS   AND    COMPLEMENTS  925 

jcctions  each  day;  four  days  after  the  last  injection  the  rabbit  blood  is  titrated  and 
usually  contains  a  fair  amount  of  anticat  hemolysin. 

2.  Heat  2  c.c.  of  the  immune  serum  at  56°  C.  for  thirty  minutes  and  inject  into 
the  external  jugular  vein  of  a  cat. 

3.  Place  the  animal  in  a  metabolic  cage  and  collect  the  urine. 

4.  After  two  or  three  days,  autopsy,  removing  the  spleen,  liver,  and  kidneys. 
Place  in  5  per  cent,  formalin  and  prepare  and  stain  sections  with  hematoxylin  and 
eosin  and  Giemsa  solution. 

(a)  Has  blood  destruction  occurred?     What  are  the  evidences? 

(b)  Would  hemolysis  occur  in  the  test-tube  with  a  heated  immune 
serum?     If  not,  why  not? 

(c)  How  then  do  you  explain  hemolysis  in  vivo  with  a  heated  serum? 

(d)  Examine  sections  of  the  spleen,  liver,  and  kidneys.     Are  there 
any  evidences  of  phagocytosis  of  blood-cells,  focal  necrosis,  and  nephritis? 
Explain  the  probable  mechanism  of  the  production  of  these  changes. 


EXERCISE  28,— AMBOCEPTORS  AND  COMPLEMENTS.    HEMOLYSINS 
EXPERIMENT  73. — TITRATION  OF  A  HEMOLYTIC  AMBOCEPTOR 

1.  Secure  1  or  2  c.c.  of  blood  from  the  ear  of  a  rabbit  which  has  been  immunized 
with  injections  of  sheep  cells.     Separate  the  serum  and  inactivate  by  heating  to  56° 
C.  for  half  an  hour.     Dilute  1: 100  by  adding  0.1  c.c.  serum  to  9.9  c.c.  normal  salt 
solution. 

2.  Prepare  40  c.c.  of  a  5  per  cent,  dilution  01  fresh  guinea-pig  serum  to  be  used  for 
complement.     Prepare  40  c.c.  of  a  2^2  per  cent,  suspension  of  washed  sheep  cells  by 
adding  1  c.c.  of  corpuscles  to  39  c.c.  of  normal  salt  solution. 

3.  Proceed  with  the  titration  as  given  in  the  text  on  page  397.     If  the  smallest 
dose,  viz.,  0.1  c.c.  of  the  1 : 100  dilution,  completely  hemolyzes  the  corpuscles,  it  will  be 
necessary  to  retitrate  with  a  dilution  of  1 : 1000. 

(a)  Is  it  necessary  to  use  exactly  the  same  amounts  of  complement 
and  corpuscles  in  all  tubes  and  if  so,  why? 

(b)  If  hemolysis  did  not  occur  at  all  in  this  experiment,  what  fac- 
tors may  be  at  fault? 

(c)  If  the  corpuscle  control  were  completely  hemolyzed,  what  de- 
duction would  you  draw? 

(d)  What  is  the  amboceptor  unit  of  this  serum? 


EXPERIMENT  74. — QUANTITATIVE  FACTORS  IN  SERUM  HEMOLYSIS 

1.  Having  determined  the  amboceptor  unit  of  the  above  antisheep  immune  serum, 
proceed  as  follows : 

2.  To  a  series  of  four  test-tubes  add  an  amount  of  immune  serum  equaling  one, 
two,  three,  and  five  amboceptor  units  respectively.     To  each  tube  add  0.5  c.c.  of  the 
1 : 20  dilution  of  complement  serum.     This  amount  is  just  half  the  dose  of  comple- 
ment used  in  titrating  the  amboceptor.     Add  1  c.c.  of  a  2^  per  cent,  suspension  of 
sheep  cells  to  each  tube  and  sufficient  salt  solution. 


926  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

3.  In  a  second  series  of  four  test-tubes  place  one-half  an  amboceptor  unit  and  the 
following  amounts  of  diluted  complement  serum:  1  c.c.,  2  c.c.,  3  c.c.,  and  4  c.c.  Add 

1  c.c.  of  the  suspension  of  sheep  corpuscles  and  sufficient  salt  solution  to  make  the 
total  volume  in  each  tube  about  equal.    Incubate  for  one  hour  and  read  the  results. 

4.  In  a  third  series  of  four  test-tubes  place  one  amboceptor  unit,  1  c.c.  of  com- 
plement serum  (1:20),  and  the  following  amounts  of  corpuscle  suspension:  1  c.c., 

2  c.c.,  3  c.c.,  and  4  c.c.   Add  sufficient  salt  solution  to  make  the  total  volume  in  each 
tube  about  equal. 

5.  Prepare  a  corpuscle  control  with  1  c.c.  of  suspension  and  4  c.c.  normal  salt 
solution. 

6.  Shake  all  tubes  gently  and  incubate  for  two  hours  at  37°  C. 

(a)  Can  an  excess  of  amboceptor  make  up  for  a  deficiency  in  comple- 
ment? 

(b)  Can  an  excess  of  complement  make  up  for  a  deficiency  of  ambo- 
ceptor? 

(c)  What  happens  when  an  excess  of  corpuscle  suspension  is  used? 

(d)  Discuss  the  importance  of  quantitative  factors  in  serum  hem- 
olysis. 

EXERCISE  29.— AMBOCEPTORS  AND  COMPLEMENTS  (Continued) 

EXPERIMENT  75. — R6LE  OF  AMBOCEPTOR  AND  COMPLEMENT  IN  HEM- 
OLYSIS 

1.  Prepare  a  2^  per  cent,  suspension  of  washed  sheep  corpuscles.     Bleed  a 
healthy  guinea-pig  under  ether  anesthesia,  separate  the  serum,  and  dilute  1 : 20  with 
normal  salt  solution  (complement).     Secure  antisheep  hemolytic  serum  (inactivated) 
whose  hemolytic  titer  is  known  (consult  instructor) . 

2.  Proceed  to  set  up  a  series  of  four  test-tubes  as  follows: 

Tube  1:  1  c.c.  corpuscle  suspension + sufficient  normal  salt  solution  to  make  the 

total  volume  about  3  c.c. 
Tube  2:  1  c.c.  corpuscle  suspension+1  c.c.  complement  serum  (1: 20)  +  sufficient 

salt  solution. 
Tube  3:  1  c.c.  corpuscle  suspension + two  hemolytic  doses  of  antisheep  hemolysin 

+  salt  solution. 
Tube  4:  1  c.c.  corpuscle  suspension+1  c.c.  complement  serum+two  doses  of 

hemolysin + salt  solution . 

3.  Shake  each  tube  gently  and  incubate  at  37°  C.  for  one  or  two  hours. 

(a)  In  which  tube  has  hemolysis  occurred?     Explain  the  results. 

(b)  What  does  inactivation  of  a  serum  mean? 

(c)  Could  some  hemolysis  occur,  using  the  complement  serum  and 
corpuscles  without  immune  hemolysin? 

EXPERIMENT  76. — SPECIFICITY  OF  AMBOCEPTORS 

1.  Prepare  a  2J^  per  cent,  suspension  of  washed  sheep  corpuscles  and  a  1  per 
cent,  suspension  of  washed  human  corpuscles.     Bleed  a  guinea-pig  under  ether, 


AMBOCEPTORS   AND    COMPLEMENTS  927 

separate  serum,  and  dilute  1 : 20  with  normal  salt  solution.     Secure  antisheep  and 
antihuman  hemolytic  serums  (inactivated)  whose  hemolytic  liters  are  known. 

2.  Proceed  as  follows: 

Tube  1:  1  c.c.  sheep  corpuscles+1  c.c.  complement  (l:20)+two  doses  of  anti- 
sheep  hemolysin+salt  solution  up  to  8  c.c. 

Tube  2:  1  c.c.  sheep  corpuscles+1  c.c.  complement  (l:20)+five  doses  of  anti- 
human  hemolysin. 

Tube  3:  1  c.c.  human  corpuscles+1  c.c.  complement  (l:20)+two  doses  of  anti- 
human  hemolysin. 

Tube  4:  1  c.c.  human  corpuscles+1  c.c.  complement  (l:20)+five  doses  of  anti- 
sheep  hemolysin. 

Tube  5:  1  c.c.  sheep  corpuscles +2  c.c.  salt  solution  (control). 

Tube  6:  1  c.c.  human  corpuscles+1  c.c.  salt  solution  (control). 

N 

3.  Shake  tubes  gently  and  incubate  for  an  hour  or  two. 

(a)  In  which  tubes  has  hemolysis  occurred? 

(b)  What  does  this  experiment  teach  as  to  the  specificity  of  these 
amboceptors? 

(c)  Discuss  the  specificity  of  hemolytic  and  bacteriolytic  ambo- 
ceptors. 

EXPERIMENT  77. — GENERAL  PROPERTIES  OF  AMBOCEPTORS 

1.  Secure  antisheep  amboceptor  so  diluted  that  1  c.c.  contains  1  hemolytic  unit. 

2.  Prepare  a  2|  per  cent,  suspension  of  sheep  corpuscles. 

3.  Secure  fresh  guinea-pig  complement  serum  and  dilute  1  :  20. 

4.  Place  two  units  of  amboceptor  into  each  of  six  test-tubes.    Place  these  in  a 
water-bath  and  heat  at  60°  C. 

5.  Remove  a  tube  from  the  water-bath  after  five  minutes,  ten  minutes,  fifteen 
minutes,  thirty  minutes,  forty-five  minutes,  and  one  hour.     After  allowing  them 
to  cool,  add  1  c.c.  corpuscle  suspension  and  1  c.c.  complement  serum  (1  : 20). 

6.  Set  up  a  tube  containing  two  units  of  amboceptor  (not  heated) +  1  c.c.  cor- 
puscle suspension +1  c.c.  complement  serum  (control).     Shake  each  tube  gently  and 
incubate  for  an  hour  at  37°  C. 

(a)  Has  the  amboceptor  deteriorated  by  exposure  to  this  degree  of 
heat? 

(b)  Discuss  the  stability  of  amboceptors  to  temperature,  age,  germi- 
cides, and  drying. 

(c)  Discuss  the  relative  stability  of  amboceptors  and  complements. 


EXERCISE  30.— AMBOCEPTORS  AND  COMPLEMENTS.— HEMOLYSINS 

(Continued) 

EXPERIMENT  78. — MECHANISM  OF  AMBOCEPTOR  ACTION 

1.  Secure  antisheep  amboceptor  so  diluted  that  1  c.c.  contains  one  hemolytic 
unit. 

2.  Prepare  a  2^  per  cent,  suspension  of  sheep  corpuscles. 

3.  Secure  fresh  guinea-pig  serum  and  dilute  1 : 20. 


928  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

4.  Into  two  centrifuge  tubes  place  four  units  of  amboceptor  and  1  c.c.  of  cor- 
puscle suspension.     Mix  and  place  one  tube  in  a  glass  of  cracked  ice,  leaving  the 
other  at  room  temperature.    After  one  hour  centrifuge  both.     Pipet  the  supernatant 
fluids  into  two  separate  test-tubes. 

5.  To  the  sedimented  corpuscles  in  both  centrifuge  tubes  add  1  c.c.  diluted  com- 
plement serum.     Mix  well.     Incubate  tubes  for  one  hour  at  37°  C. 

6.  To  the  supernatant  fluid  of  each  tube  add  1  c.c.  corpuscle  suspension  and  1 
c.c.  of  diluted  complement  serum.     Mix.     Incubate  tubes  for  an  hour. 

(a)  In  which  tubes  has  hemolysis  occurred? 

(b)  How  do  you  explain  the  results? 

(c)  Discuss  the  prevailing  views  of  amboceptor  action. 

EXPERIMENT  79. — A  FURTHER  STUDY  OF  THE  MECHANISM  OF  AMBO- 
CEPTORS 

1.  In  a  centrifuge  tube  place  two  hemolytic  doses  of  antisheep  hemolysin  and  2  c.c. 
of  fresh  guinea-pig  complement  diluted  1  :  20.  Place  in  a  glass  of  cracked  ice  until  the 
mixture  is  thoroughly  chilled.    Then  add  2  c.c.  of  a  1\  per  cent,  suspension  of  sheep 
cells  (also  chilled).     Mix  and  keep  at  a  low  temperature  for  an  hour,  centrifuge 
thoroughly,  and  pipet  the  supernatant  fluid  to  a  separate  test-tube. 

2.  In  a  second  centrifuge  tube  place  2  c.c.  of  diluted  complement  and  2  c.c.  cor- 
puscle suspension.     Keep  at  room  temperature  for  one  hour,  centrifuge,  and  pipet  the 
supernatant  fluid  into  a  test-tube. 

3.  Proceed  as  follows:  To  2  c.c.  of  the  supernatant  fluid  from  the  first  centrifuge 
tube  add  1  c.c.  corpuscle  suspension;  to  the  remaining  2  c.c.  add  1  c.c.  corpuscle  sus- 
pension and  two  units  of  amboceptor;   to  the  sedimented  corpuscles  add  2  c.c.  of 
diluted  complement  serum. 

4.  To  2  c.c.  of  the  supernatant  fluid  from  the  second  centrifuge  tube  add  1  c.c. 
corpuscle  suspension;  to  the  remaining  2  c.c.  add  1  c.c.  corpuscle  suspension  and  two 
units  of  hemolytic  amboceptor. 

5.  To  the  sedimented  corpuscles  in  both  centrifuge  tubes  add  1  c.c.  complement 
serum  and  1  c.c.  salt  solution.     Mix  well. 

6.  Shake  all  tubes  gently  and  incubate  for  one  hour  at  37°  C. 

(a)  In  which  tubes  has  hemolysis  occurred? 

(b)  Does  complement  unite  directly  with  corpuscles? 

(c)  What  evidence  have  you  that  amboceptors  unite  directly  with 
corpuscles? 

(d)  What  is  meant  by  the  term  " sensitizing  corpuscles"? 

(e)  Is  complement-amboceptor  activity  apparent  at  low  temperature? 

(f)  What  temperature  best  favors  complement-amboceptor  activity? 

EXERCISE  3J.— AMBOCEPTORS  AND  COMPLEMENTS.— HEMOLYSINS 

(Continued) 

EXPERIMENT    80 — NATURAL  HEMOLYSINS.    REMOVAL    OF    NATURAL 
HEMOLYSINS 

1.  Secure  1  c.c.  of  serum  from  the  blood  of  five  different  persons  and  inactivate 
in  the  water-bath. 


AMBOCEPTORS   AND    COMPLEMENTS  929 

2.  Remove  0.5  c.c.  of  serum  from  each  to  five  separate  centrifuge  tubes  and  add 
4.5  c.c.  of  2}^  per  cent,  suspension  of  sheep  cells  to  each.     Shake  gently  and  after 
half  an  hour  at  room  temperature  centrifuge  thoroughly  and  pipet  the  supernatant 
dilute  serum  to  separate  tubes.     Do  not  discard  the  corpuscles  in  the  centrifuge 
tubes. 

3.  To  each  of  the  remaining  0.5  c.c.  amounts  of  serum  add  4.5  c.c.  salt  solution 
and  place  1  c.c.  and  2  c.c.  into  two  test-tubes  respectively  (0.1  c.c.  and  0.2  c.c.  undi- 
luted serum). 

4.  Into  two  more  test-tubes  place  1  and  2  c.c.  respectively  of  the  diluted  serum 
which  has  been  treated  with  corpuscles. 

5.  Add  to  each  tube  1  c.c.  of  guinea-pig  complement  (1:20),  1  c.c.  of  2^  per 
cent,  suspension  of  sheep  cells,  and  sufficient  salt  solution.     Shake  gently  and  incu- 
bate for  one  hour. 

6.  To  the  sedimented  corpuscles  in  the  centrifuge  tubes  add  2  c.c.  of  diluted  com- 
plement serum  and  sufficient  salt  solution.     Shake  gently  and  incubate  for  one  hour. 

(a)  Has  hemolysis  occurred  with  the  untreated  serums? 

(b)  How  do  you  explain  the  results? 

(c)  Has  hemolysis  occurred  with  the  treated  serums  and  if  not,  why 
not? 

(d)  Has  hemolysis  occurred  with  the  corpuscles  used  in  treating 
the  serums  and  why? 

(e)  Of  what  practical  importance  are  natural  hemolysins? 

(f)  Is  natural  antihuman  hemolysin  ever  found  in  human  serums? 
What  are  they  called? 

Note. — The  quantity  of  natural  amboceptor  in  any  of  these  serums 
may  be  determined  by  the  method  of  titration  given  in  the  text.  In 
choosing  five  serums  at  random  it  may  be  possible  that  some  will  not 
show  an  appreciable  amount  of  natural  antisheep  amboceptor. 


EXERCISE  32.— AMBOCEPTORS  AND  COMPLEMENTS  (Continued) 
EXPERIMENT  81. — HEMOLYTIC  COMPLEMENT 

1.  After  giving  a  rabbit  three  intravenous  injections  of  5  c.c.  of  a  10  per  cent, 
suspension  of  washed  sheep  cells  at  intervals  of  three  days,  remove  three  cubic  centi- 
meters of  blood  from  an  ear  and  separate  the  serum.     Dilute  the  serum  1 : 10  with 
salt  solution. 

2.  Into  four  test-tubes  place  1  c.c.  of  a  2^  per  cent,  suspension  of  sheep  corpuscles 
and  increasing  amounts  of  the  above  dilution  of  rabbit  serum  (must  be  fresh — not 
over  twelve  hours  old)  as  follows:  0.4,  0.8,  1,  and  2  c.c.;  add  sufficient  salt  solution. 
Shake  and  incubate  for  one  hour. 

(a)  Has  hemolysis  occurred? 

(b)  How  do  you  explain  the  reaction? 

(c)  From  where  was  the  complement  derived? 

(d)  Are  complements  found  in  the  bloods  of  all  animals? 

(e)  Discuss  the  question  of  the  multiplicity  of  complements. 

59 


930  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

EXPERIMENT  82. — INACTIVATION  AND  REACTIVATION  OF  COMPLEMENT 

1.  Heat  1  c.c.  of  the  1: 10  dilution  of  fresh  rabbit  immune  serum  used  in  the  pre- 
ceding experiment  to  56°  C.  for  half  an  hour  (in  a  water-bath). 

2.  In  a  test-tube  place  a  cubic  centimeter  of  this  heated  serum  and  1  c.c.  of  a  2^ 
per  cent,  suspension  of  sheep  cells  and  sufficient  salt  solution.     Shake  gently  and 
incubate  for  one  hour. 

(a)  Does  hemolysis  occur? 

(b)  How  do  you  explain  the  result? 

(c)  What  is  meant  by  inactivation  of  complement? 

(d)  Is  complement  thermostabile? 

3.  Add  to  the  tube  1  c.c.  of  fresh  guinea-pig  serum  diluted  1 : 10  and  reincubate 
for  an  hour. 

(e)  Has  hemolysis  occurred? 

(f)  How  do  you  explain  the  result? 

(g)  What  is  meant  by  reinactivation? 

EXPERIMENT  83. — GENERAL  PROPERTIES  OF  COMPLEMENT 

1.  Secure  antisheep  amboceptor  the  hemolytic  unit  of  which  is  known  by  pre- 
vious titration. 

2.  Prepare  a  2^  per  cent,  suspension  of  washed  sheep  corpuscles. 

3.  Into  a  series  of  four  test-tubes  place  the  following: 
Tube  1 :  1  c.c.  of  fresh  guinea-pig  serum  diluted  1 : 20. 

Tube  2:  1  c.c.  of  guinea-pig  serum  (1:20)  which  has  been  kept  at  room  tempera- 
ture for  two  days. 

Tube  3:  1  c.c.  of  guinea-pig  serum  (1:20)  three  days  old. 

Tube  4:  1  c.c.  of  guinea-pig  serum  (1:20)  five  days  old. 

Add  1  c.c.  of  a  2^  per  cent,  suspension  of  sheep  corpuscles,  two  hemolytic  units 
of  antisheep  amboceptor,  and  sufficient  normal  salt  solution  to  each  tube.  Shake 
gently  and  incubate  at  37°  C.  for  one  hour. 

4.  Into  a  series  of  six  test-tubes  place  1  c.c.  of  fresh  guinea-pig  complement  serum 
diluted  1 : 20.     Place  these  in  a  water-bath  at  56°  C.    At  intervals  of  ten,  twenty, 
thirty,  forty,  fifty,  and  sixty  minutes  remove  a  tube,  add  1  c.c.  corpuscle  suspension, 
two  units  of  amboceptor,  and  sufficient  salt  solution.     Shake  gently  each  tube  and 
incubate  for  one  hour  at  37°  C. 

(a)  Record  the  results.     Does  hemolytic  complement  deteriorate 
readily  at  room  temperature? 

(b)  What  are  complementoids? 

(c)  What  practical   significance  has  this   experiment?     Should   a 
complement  serum  be  fresh  when  used  in  hemolytic  work? 

(d)  Is  complement  thermolabile?     How  long  does  it  take  to  destroy 
a  diluted  complement  at  56°  C.? 

(e)  In  inactivating  a  serum  why  do  we  not  use  a  higher  temperature? 


AMBOCEPTORS   AND    COMPLEMENTS  931 

EXERCISE  33.— AMBOCEPTORS  AND  COMPLEMENTS  (Continued) 
EXPERIMENT  84. — TITRATION  OF  HEMOLYTIC  COMPLEMENT 

1.  Prepare  20  c.c.  of  complement  1:20;  prepare  a  2J^  per  cent,  suspension  of 
sheep  cells. 

2.  To  a  series  of  10  test-tubes  add  increasing  doses  of  diluted  complement  serum: 
0.1,  0.2,  0.4,  0.5,  0.6,  0.7,  0.8,  0.9,  1,  and  1.5  c.c.;  add  1^  units  of  hemolytic  ambo- 
ceptor  (determined  by  previous  titration) ;  1  c.c.  of  corpuscle  suspension  and  sufficient 
salt  solution  to  bring  the  total  volume  in  each  tube  to  3  c.c. 

3.  Shake  gently  and  incubate  for  one  hour  at  37°  C. 

(a)  Record  the  results.     What  is  the  complement  unit  of  this  serum? 

(b)  What  animal  serum  is  best  adapted  for  complement  in  hemolytic 
work? 

(c)  Is  the  complement  content  of  the  serums  of  different  animals 
constant?      Does  it  vary  in  animals  of  the  same  species?     In  the  same 
animal  at  different  times? 

EXPERIMENT  85. — PHENOMENON  OF  COMPLEMENT  FIXATION 

This  experiment  is  introduced  here  to  show  the  Bordet-Gengou 
phenomenon  of  complement  fixation.  The  exact  technic  of  complement- 
fixation  reactions  as  conducted  for  the  diagnosis  of  syphilis  and  other 
infections  will  be  given  in  subsequent  exercises. 

1.  Use  the  same  complement  serum,  amboceptor  and  corpuscle  suspension  as 
used  in  the  preceding  experiment.     The  unit  of  complement  is  now  known. 

2.  Secure  1  c.c.  of  an  antigonococcus  and  a  normal  serum  and  heat  at  56°  C.  for 
thirty  minutes. 

3.  Secure  an  emulsion  of  gonococci  which  is  now  called  the  antigen;  for  the  proper 
dose  to  use  consult  the  instructor. 

4.  Proceed  as  follows: 

Tube  1 :  0.2  c.c.  antigonococcus  serum  -f-  dose  of  antigen  +  2  units  of  comple- 
ment +  normal  salt  solution. 

Tube  2:  0.2  c.c.  antigonococcus  serum  +  2  units  of  complement  -f-  normal  salt 
solution  (serum  control). 

Tube  3:  0.2  c.c.  normal  serum  +  doses  of  antigen  +  2  units  of  complement  + 
normal  salt  solution. 

Tube  4:  0.2  c.c.  normal  serum  +  2  units  of  complement  +  normal  salt  solution 
(serum  control). 

Tube  5 :  dose  of  antigen  +  2  units  of  complement  +  normal  salt  solution  (anti- 
gen control). 

Tube  6:  2  units  of  complement  +  normal  salt  solution  (hemolytic  control). 

Tube  7:  1  c.c.  corpuscle  suspension  +  normal  salt  solution  (corpuscle  control). 

Plug  tube  with  cotton  as  it  is  finished. 

5.  Shake  all  tubes  gently  and  incubate  for  one  hour  at  37°  C. 

6.  Add  1^  units  of  amboceptor  and  1  c.c.  corpuscle  suspension  to  all  tubes 
except  corpuscle  control.     Shake  gently  and  reincubate  for  one  hour. 


932  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

(a)  Examine  tubes  and  record  your  results. 

(b)  Why  was  the  serum  control  on  each  serum  necessary? 

(c)  Why  was  the  hemolytic  system  control  necessary? 

(d)  Why  was  the  antigen  control  necessary? 

(e)  What  is  meant  by  non-specific  complement  fixation? 

(f)  What  is  meant  by  specific  complement  fixation?     Explain  the 
phenomenon.     Which  tube  of  this  series  shows  specific  complement  fixa- 
tion and  why?     Is  there  any  evidence  of  non-specific  fixation  in  any  of 
the  tubes?     If  so,  what  bearing  would  this  have  on  the  results  of  this 
test? 

(g)  What  is  meant  by  complement  deviation? 

(h)  Upon  what  does  the  specificity  of  complement  fixation  depend? 

EXERCISE  34.— ANTIGENS  FOR  THE  WASSERMANN  REACTIONS 

EXPERIMENT  86. — PREPARATION  OF  ANTIGENS  FOR  THE  WASSERMANN 
REACTION 

1.  Prepare  50  c.c.  of  an  alcoholic  extract  of  syphilitic  liver  (see  page  444). 

2.  Prepare  an  extract  of  acetone-insoluble  lipoids  from  20  grams  of  beef  heart 
(see  page  446). 

3.  Prepare  50  c.c.  of  a  cholesterinized  alcoholic  extract  of  human  heart  (see 
page  421). 

(a)  Are  antigens  for  the  Wassermann  syphilis  reaction  specific? 

(b)  Upon  what  does  the  high  specificity  of  the  Wassermann  reac- 
tion depend? 

(c)  What  constitutes  a  specific  antigen  for  the  Wassermann  reac- 
tion? 

(d)  Discuss  the  important  relation  of  antigen  to  the  Wassermann  re- 
action. 

EXERCISE  35.— ANTIGENS  (Continued) 
EXPERIMENT  87. — METHODS  OF  TITRATION  OF  ANTIGENS 

While  the  above  extracts  are  in  the  course  of  preparation,  stock  ex- 
tracts may  be  used  for  titration.  After  the  student  has  finished  the 
above  three  extracts,  they  are  titrated  in  a  similar  manner. 

1.  Dilute  1  c.c.  of  an  alcoholic  extract  of  syphilitic  liver  in  a  test-tube  1:10  by 
slowly  adding  9  c.c.  of  normal  saline  solution.     Dilute  a  cholesterinized  extract  (1 : 20) 
by  slowly  adding  9.5  c.c.  salt  solution  to  0.5  c.c.  of  extract. 

2.  Conduct  the  anticomplementary  titration  of  each  (page  452) . 

3.  Conduct  the  antigenic  titration  of  each  (page  455) . 

(a)  What  is  meant  by  the  anticomplementary  dose  of  an  antigen? 

(b)  What  is  meant  by  the  antigenic  dose  of  antigen? 


WASSERMANN    REACTION  933 

(c)  Why  are  these  titrations  so  important? 

(d)  In  a  complement-fixation  test  what  would  be  the  result  if  the 
antigen  were  used  in  the  anticomplementary  dose?     What  would  be 
the  result  if  the  antigen  were  used  in  less  than  the  antigenic  dose? 

(e)  What  constitutes  a  satisfactory  antigen  for  any  complement-fix- 
ation test? 

(f )  In  conducting  a  diagnostic  reaction,  should  the  antigen  be  used 
in  exactly  one  antigenic  dose  or  double  or  treble  this  amount?     Why 
and  under  what  conditions  would  this  be  a  safe  procedure? 

(g)  Why  should  the  antigen  be  diluted  slowly  with  salt  solution? 
(h)  Why  is  a  serum  control  so  important  in  these  titrations?    What 

other  controls  are  used  and  why? 

(i)  Which  is  more  important,  the  anticomplementary  or  antigenic 
titration  and  why? 

EXERCISE  36.— WASSERMANN  REACTION 
EXPERIMENT  88. — ANTICOMPLEMENTARY  ACTION  OF  SERUMS 

1.  Secure  1  c.c.  of  five  different  specimens  of  human  serums  which  have  been 
standing  in  the  laboratory  for  one,  two,  four,  six,  and  ten  days  respectively.     The 
last  specimen  should  be  intentionally  infected  with  a  culture  of  staphy  loco  ecus. 

2.  Divide  each  serum  into  two  portions  and  heat  portion  "B"  to  56°  C.  for  half 
an  hour. 

3.  Place  0.2  c.c.  of  each  specimen  (unheated)  in  a  series  of  five  test-tubes. 

4.  Place  0.2  c.c.  of  each  specimen  (heated)  in  a  second  series  of  five  test-tubes. 

5.  To  each  tube  add  1  c.c.  of  complement  1:20  and  sufficient  salt  solution  to 
bring  the  total  volume  to  3  c.c.     Shake  gently  and  incubate  for  half  an  hour.     Add 
1^  units  of  amboceptor  and  1  c.c.  corpuscle  suspension  to  each  tube  and  incubate 
for  one  hour. 

6.  A  hemolytic  system  control  (complement,  corpuscles,  and  amboceptor)  should 
be  included;  also  a  corpuscle  control. 

(a)  Record  your  results.     Are  any  of  the  serums  anticomplementary? 
How  do  you  determine  this? 

(b)  What  is  meant  by  the  terms  anticomplementary  action  of  a  serum? 

(c)  What  significance  has  this  phenomenon  in  complement-fixation 
work? 

(d)  Are  anticomplementary  bodies  thermolabile  or  thermostabile? 
May  both  exist?     Under  what  conditions  are  each  most  likely  to  be 
present? 

(e)  How  are  thermolabile  anticomplementary  bodies  removed  or 
their  influence  overcome?     When  a  serum  is  three  or  more  days  old, 
what  is  the  chief  object  of  heating  it  before  it  is  used  in  a  complement- 
fixation  test? 


934  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

(f)  Does  complement  keep  for  three  days? 

(g)  Is  it  possible  to  remove  the  thermostabile  anticomplementary 
action  of  a  serum?     Could  such  a  serum  be  used  in  a  complement-fixa- 
tion reaction?     How  is  this  condition  of  the  serum  detected? 

(h)  Under  what  conditions  is  a  serum  likely  to  become  anticomple- 
mentary in  action?  Is  a  sterile  serum  likely  to  become  anticomple- 
mentary? 

EXERCISE  37.— "WASSERMANN  REACTION  (Continued) 
EXPERIMENT  89. — WASSERMANN  REACTION  (FIRST  METHOD) 

1.  Secure  four  specimens  of  blood:    one  from  a  known  syphilitic  person;    the 
second  from  a  known  normal  person,  the  third  and  fourth  specimens  for  diagnosis. 
Also  a  specimen  of  cerebrospinal  fluid. 

2.  Conduct   a  Wassermann  reaction  with  each  serum  after  the  first  method, 
using  an  antigen  of  alcoholic  extract  of  syphilitic  liver. 

(a)  Record  your  results.     In  case  the  hemolytic  system  control  was 
not  completely  hemolyzed,  what  may  be  the  reasons  and  what  influence 
would  this  result  have  on  interpreting  the  reactions? 

(b)  In  case  the  antigen  control  was  not  completely  hemolyzed,  what 
influence  would  this  result  have  on  the  reactions? 

(c)  If  a  serum  control  were  incompletely  hemolyzed,  what  influence 
would  this  have  on  the  results  of  the  test? 

(d)  Is  this  method  a  quantitative  reaction? 

(e)  What  is  the  nature  of  the  antibody  in  a  syphilitic  serum? 

(f)  How  are  these  reactions  recorded?     How  should  they  be   re- 
ported to  a  clinician? 

(g)  Discuss  the  value  of  the  Wassermann  reaction  in  the  various 
stages  of  syphilis  as  a  guide  to  treatment. 

EXERCISE  38,— WASSERMANN  REACTION  (Continued) 
EXPERIMENT  90. — WASSERMANN  REACTION  (SECOND  METHOD) 

1.  Conduct  a  Wassermann  reaction  with  each  of  five  specimens  of  serum  after 
the  second  method,  using  an  alcoholic  extract  of  syphilitic  liver,  an  extract  of  acetone- 
insoluble  lipoids,  and  an  alcoholic  extract  of  heart  reenforced  with  cholesterin.  One 
of  these  serums  should  be  from  a  syphilitic  person  (positive  control)  and  one  from  a 
normal  person  (negative  control) . 

(a)  What  are  the  advantages  of  using  more  than  one  antigen? 

(b)  Discuss  the  relative  value  of  these  three  antigens. 

(c)  Under  what  conditions  may  a  cholesterinized  extract  be  used? 


NOGUCHI   MODIFICATION   OF  THE   WASSERMANN   REACTION        935 

EXERCISE  39.— WASSERMANN  REACTION  (Continued) 
EXPERIMENT  91.— WASSERMANN  REACTION  (THIRD  METHOD) 

Conduct  a  Wassermann  reaction  with  each  of  four  specimens  of  serum  after 
the  third  method,  using  an  extract  of  acetone-insoluble  lipoids.  One  of  these  serums 
should  be  a  positive  control  and  one  a  normal  or  negative  control. 

What  is  the  advantage  of  using  a  quantitative  test? 

EXERCISE  40.— WASSERMANN  REACTION  (Continued) 
EXPERIMENT  92. — WASSERMANN  REACTION  (FOURTH  METHOD) 

Conduct  a  Wassermann  reaction  with  a  known  positive,  a  known  negative, 
and  two  unknown  serums  after  the  fourth  method,  using  an  extract  of  aceton-insoluble 
lipoids. 

What  are  the  main  advantages  of  this  technic? 


EXERCISE  4*.— NOGUCHI  MODIFICATION  OF  THE  WASSERMANN  RE- 
ACTION 

EXPERIMENT  93. — TITRATION  OF  ANTIHUMAN  HEMOLYSIN 

1.  Secure  0.1  c.c.  of  inactivated  antihuman  rabbit  amboceptor  serum  and  dilute 
1:100  by  adding  9.9  c.c.  normal  saline  solution. 

2.  To  a  series  of  six  small  test-tubes  add  increasing  amounts  of  diluted  ambo- 
ceptor as  follows:  0.1,  0.2,  0.4,  0.6,  0.8,  and  1  c.c.,  add  0.1  c.c.  of  40  per  cent,  com- 
plement, 1  c.c.  of  1  per  cent,  human  corpuscle  suspension,  and  sufficient  saline  solu- 
tion to  bring  the  total  volume  to  2  c.c. 

3.  Incubate  for  one  to  two  hours,  shaking  the  tubes  once  or  twice  during  this 
time. 

(a)  Are  there  any  evidences  of  hemagglutination? 

(b)  What  constitutes  the  unit  of  hemolytic  amboceptor? 

(c)  Is  an  antihuman  hemolytic  system  more  delicate  than  an  anti- 
sheep  system  in  complement-fixation  work? 

(d)  What  are  the  advantages  of  using  an  antihuman  hemolytic  sys- 
tem? 

EXPERIMENT  94. — TECHNIC  OF  THE  NOGUCHI  MODIFICATION 

1.  Secure  five  specimens  of  blood  not  over  twenty-four  hours  old,  including  at 
least  one  positive  and  one  negative  serum. 

2.  Conduct  the  Noguchi  reaction  with  each  serum  in  the  unheated  or  active 
state,  using  an  antigen  of  acetone-insoluble  lipoids.     (See  page  475.) 

3.  Conduct  the  reactions  with  the  same  antigen,  using  the  serums  after  heating 
to  56°  C.  for  half  an  hour. 

(a)  Record  your  results.     Are  they  equal  in  both  series? 

(b)  What  is  meant  by  proteotropic  reaction? 

(c)  What  effect  has  heat  upon  syphilis  reagin? 


936  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

(d)  Does  an  active  serum  react  more  delicately  than  an  inactivated 
one? 

(e)  In  performing  this  test  with  unheated  serum  what  precautions 
are  to  be  observed? 

EXERCISE  42.— WASSERMANN  AND  NOGUCHI  REACTIONS 
EXPERIMENT  95. — COMPARISON  OF  METHODS 

1.  Secure  five  serums  and  a  cerebrospinal  fluid. 

2.  Conduct  a  Wassermann  reaction  with  each  after  the  second  method. 

3.  Conduct  a  Noguchi  test  with  each  serum  inactivated  and  using  an  extract  of 
acetone-insoluble  lipoids. 

(a)  How  do  the  results  compare? 

(b)  Which  reactions  are  more  easily  read? 

(c)  Discuss  the  relative  delicacy  and  value  of  the  Wassermann  and 
Noguchi  reactions  with  both  active  and  inactivated  serum  in  the  latter. 

EXERCISE  43,— GONOCOCCUS  COMPLEMENT-FIXATION  REACTION 
EXPERIMENT  96. — TITRATION  OF  GONOCOCCUS  ANTIGEN 

1.  Secure  1  c.c.  of  gonococcus  antigen  and  dilute  1:10  by  adding  9  c.c.  normal 
saline  solution. 

2.  Secure  1  c.c.  of  an  antigonococcus  serum  or  the  serum  of  a  person  who  reacts 
positively  and  inactivate. 

3.  Conduct  an  anticomplementary  and  antigenic  titration  as  described  on  page 
506. 

4.  A  similar  titration  may  be  conducted  with  glanders  antigen  and  antiserum. 

(a)  Give  three  methods  of  preparing  a  bacterial  antigen. 

(b)  Is  it  advisable  to  make  polyvalent  antigens  and  whjr? 

(c)  Which  is  of  more  practical  value,  the  anticomplementary  or  anti- 
genic titration  and  why? 

(d)  What  part  of  the  anticomplementary  dose  of  an  antigen  may  be 
safely  used  in  a  diagnostic  test? 

(e)  Is  it  advisable  to  titrate  a  bacterial  antigen  at  frequent  intervals? 

EXERCISE  44.— GONOCOCCUS  COMPLEMENT-FIXATION  REACTION 
EXPERIMENT  97. — TECHNIC  OF  THE  GONOCOCCUS  REACTION 

1.  Secure  six  specimens  of  serum  from  a  genito-urinary  clinic,  particularly  of 
men  suffering  with  chronic  gonococcus  infections.     Also  a  known  positive  and  a 
known  normal  serum  for  controls. 

2.  Conduct  the  reactions  after  the  method  described  on  page  504. 

(a)  Is  the  gonococcus  reaction  specific? 


COMPLEMENT    FIXATION    IN   THE    DIFFERENTIATION    OF    PROTEINS   937 

(b)  Is  complement  fixation  in  bacterial  infections  likely  to  be  as  well 
marked  as  in  syphilis? 

(c)  In  what  cases  of  gonococcus  infection  is  the  reaction 'likely  to  be 
negative?     Likely  to  be  positive? 

(d)  How  soon  after  infection  is  the  reaction  likely  to  become  posi- 
tive? 

(e)  Discuss  the  practical  value  of  the  gonococcus  complement-fixation 
test. 

EXERCISE  45.— GONOCOCCUS  COMPLEMENT-FIXATION  TEST 
EXPERIMENT  98. — TECHNIC  OF  THE  GONOCOCCUS  REACTION 

1.  Using  a  similar  set  of  serums  as  in  the  preceding  experiment,  conduct  the 
reactions  with  the  one-tenth  technic  as  described  on  page  508. 

(a)  What  is  the  main  advantage  of  this  technic? 

(b)  By  which  method  are  the  reactions  more  easily  read  and  re- 
corded? 

EXERCISE  46.— COMPLEMENT  FIXATION  IN  THE  DIFFERENTIATION  OF 

PROTEINS 

EXPERIMENT  99. — TITRATION  OF  IMMUNE  SERUMS 

1.  Secure  1  c.c.  each  of  antihuman  and  antihorse  serum.     Inactivate  both. 

2.  Secure  0.1  c.c.  each  of  fresh  human  and  horse  serum  and  dilute  1: 1000. 

3.  Conduct  an  antigenic  titration  of  each  immune  serum  as  described  on  page  528. 

(a)  Explain  the  mechanism  of  this  reaction. 

(b)  Are  protein  amboceptors  specific? 

(c)  Discuss  the  question  of  inhibition  of  hemolysis  due  to  a  precipitin 
reaction. 


EXERCISE  47.— COMPLEMENT  FIXATION  IN  THE  DIFFERENTIATION  OF 

PROTEINS 

EXPERIMENT  100. — TECHNIC  OF  THE  FORENSIC  BLOOD  TEST 

1.  Secure  from  the  instructor  two  pieces  of  gauze  stained  respectively  with  human 
and  horse  blood.     These  are  to  be  numbered  and  their  identity  known  only  to  in- 
structor. 

2.  Secure  1  c.c.  each  of  antihuman  and  antihorse  serum. 

3.  Secure  0.1  c.c.  of  known  human  and  horse  serum  for  controls. 

4.  Proceed  with  the  test  for  identifying  these  bloods  as  described  on  page  527. 

(a)  Discuss  the  specificity  of  this  test. 

(b)  Is  this  test  more  delicate  than  the  precipitin  reaction? 


938  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

(c)  Which  is  the  more  liable  to  error  in  technic? 

(d)  Discuss  the  applicability  of  the  complement-fixation  technic  to 
the  differentiation  of  proteins  in  general,  as  animal,  vegetable,  and  bac- 
terial proteins. 


EXERCISE  48.— VENOM  HEMOLYSIS 
EXPERIMENT  101. — VENOM  HEMOLYSIS  IN  SYPHILIS 

1.  Secure  1  c.c.  of  venom  solution  1: 1000. 

2.  Secure  four  specimens  of  blood  (for  technic  see  page  408)  from  cases  of  late 
secondary  or  tertiary  syphilis  and  one  specimen  of  normal  blood.     Also  a  known 
normal  blood  for  a  control. 

3.  Prepare  the  subdilutions  of  venom  and  conduct  the  tests  after  the  technic 
described  on  page  409. 

(a)  Discuss  the  prevailing  views  regarding  the  mechanism  of  venom 
hemolysis. 

(b)  Discuss  the  value  of  the  venom  test  in  the  diagnosis  of  syphilis. 

(c)  Discuss  the  main  points  in  the  technic. 

EXERCISE  49.— BACTERIOLYSIS 
EXPERIMENT  102. — PFEIFFER  BACTERIOLYTIC  TEST 

1.  Secure  1  c.c.  of  serum  from  a  rabbit  which  has  been  immunized  with  typhoid 
bacilli.     By  working  with  cholera  and  a  highly  potent  serum  better  results  are  secured, 
but  as  there  is  probably  more  danger  connected  with  the  handling  of  cholera  cultures, 
typhoid  may  be  substituted  with  fairly  good  results.     Inactivate  by  heating  to  56° 
C.  for  half  an  hour. 

2.  Prepare  a  twenty-four-hour-old  agar  culture  of  a  suitable  strain  of  Bacillus 
typhosus. 

3.  Prepare  a  1 : 100  dilution  of  the  immune  serum  and  place  3  c.c.  in  a  small  test- 
tube.     Add  three  loopfuls  of  culture  and  emulsify  thoroughly.     Inject  a  guinea-pig 
intraperitoneally  with  2  c.c.  of  the  emulsion. 

4.  At  intervals  of  ten,  twenty,  forty,  and  sixty  minutes  withdraw  small  amounts 
of  exudate  and  study  bacteriolysis;  prepare  hanging-drop  preparations  which  may  be 
compared  with  a  similar  control  on  the  culture;   prepare  smears  of  the  culture  and 
peritoneal  exudate  and  stain  with  carbol-thionin  or  carbolfuchsin. 

5.  If  desirable,  the  bacteriolytic  titer  of  the  serum  may  be  determined  after  the 
method  given  in  the  text. 

(a)  Describe  the  phenomenon  of  bacteriolysis. 

(b)  Are  bacteriolysis  and  hemolysis   similar  processes?     Give  the 
source  of  complement  in  this  reaction. 

(c)  Discuss  the  specificity  of  bacteriolytic  reactions. 

(d)  Discuss  the  practical  value  of  the  bacteriolytic  test  in  the  diag- 
nosis of  a  microorganism. 


BACTERIOLYSIS  939 

EXERCISE  50.— BACTERIOLYSIS 

EXPERIMENT   103. — MICROSCOPIC  METHOD  OF  MEASURING  THE  BAC- 
TERIOLYTIC  POWER  OF  THE  BLOOD 

This  method  may  be  employed  for  a  rapid  estimation  of  the  bac- 
teriolysin  produced  in  the  blood  in  response  to  an  inoculation  of  typhoid 
vaccine. 

1.  Secure  a  small  quantity  of  the  patient's  serum  by  collecting  blood  aseptically 
in  a  Wright  capsule.     About  1  c.c.  of  serum  will  be  sufficient.     Secure  a  control 
serum,  preferably  a  "pooled  serum,"  in  the  same  manner.     Prepare  dilutions  of  the 
patient's  serum  in  the  following  manner:  Place  a  series  of  six  small  test-tubes  (sterile) 
in  a  rack;  add  1  c.c.  sterile  broth  to  each  tube;  into  the  first  tube  place  1  c.c.  of  the 
fresh  serum  from  the  patient  (1:2  dilution) ;  mix  well  and  transfer  1  c.c.  to  the  second 
tube;  mix  and  transfer  1  c.c.  to  the  third,  and  so  on  to  the  last  tube,  when  1  c.c.  is 
discarded. 

2.  Secure  a  twenty-four-hour  culture  of  typhoid  bacilli  in  neutral  bouillon. 

3.  Take  a  simple  capillary  pipet  with  a  long  stem,  plugged  with  cotton  and  steril- 
ized, and  make  a  mark  about  3  cm.  from  the  end.     Draw  up  the  highest  dilution  of 
serum  to  the  pencil  mark,  then  a  bubble  of  air,  and  finally  an  equal  volume  of  culture. 
Thoroughly  mix  by  carefully  aspirating  and  driving  out  the  contents  on  a  hollow 
ground  slide  or  in  a  small  tubule.     Aspirate  the  mixture  into  the  middle  portion  of 
the  stem  and  seal  the  pipet  in  a  flame.     Label  with  the  final  dilution  (1 : 128). 

4.  Proceed  in  the  same  manner  with  the  remaining  five  dilutions  of  the  patient's 
serum,  which  then,  mixed  with  an  equal  quantity  of  culture,  equals  1 : 64,  1 : 32,  1 : 16, 
1 : 8,    and  1 : 4.     Place  these  pipets  in  a  large  test-tube  and  label  with  the  patient's 
name  and  time  when  placed  in  the  incubator. 

5.  Prepare  a  similar  set  of  pipets  using  the  control  serum. 

6.  Prepare  a  culture  control  by  mixing  equal  volumes  of  culture  and  sterile  broth. 

7.  Place  all  pipets  in  the  incubator  at  37°  C.  for  two  hours. 

8.  Prepare  hanging-drop  preparations  of  each  pipet  by  breaking  off  the  tip  and 
placing  a  drop  of  the  contents  (after  mixing)  on  a  cover  slide  and  suspending  in  the 
usual  manner.     First  examine  the  culture  control  and  then  each  of  the  various  dilu- 
tions, noting  in  each  case  the  dilution  in  which  there  is  the  first  bacteriolytic  effect, 
and  the  dilution  in  which  there  is  a  complete  effect;   arrive  at  the  bacteriolytic  index 
by  comparing  the  patient's  and  the  control  blood  just  as  the  opsonic  index  is  stated. 

(a)  What  are  the  first  evidences  of  bacteriolysis? 

(b)  Are  endotoxins  neutralized  when  bacteriolysis  occurs? 


EXERCISE  5f.— BACTERIOLYSIS 

EXPERIMENT   104. — METHOD  OF  MEASURING  THE  BACTERICIDAL  AC- 
TIVITY OF  THE  BLOOD  IN  VITRO  (METHOD  OF  STERN  AND  KORTE) 

With  a  culture  of  typhoid  bacilli,  rabbit  typhoid  immune  serum,  and   rabbit 
complement  serum,  a  test  may  be  carried  out  after  the  method  given  in  the  text. 

(a)  What  main  precautions  are  to  be  followed  in  this  method? 

(b)  Discuss  complement  deviation. 

(c)  Discuss  the  practical  value  of  this  method. 


940  EXPERIMENTAL    INFECTION    AND    IMMUNITY 

EXERCISE  52.— CYTOTOXINS 
EXPERIMENT  105. — ACTION  OF  NEPHROTOXIN 

1.  Prepare  an  emulsion  of  dog  kidney  as  described  on  page  74  and  immunize 
arabbit. 

2.  Inject  a  dog  intravenously  with  2  c.c.  of  rabbit  immune  serum  per  kilo  of  body 
weight. 

3.  Place  the  animal  in  a  metabolism  cage,  collect  all  urine,  and  carefully  examine 
for  albumin,  casts,  and  hemoglobin;  make  quantitative  albumin  determinations. 

4.  After  three  days  autopsy  and  examine  the  kidneys  and  liver  histologically. 

5.  Heat  the  immune  serum  to  56°  C.  for  thirty  minutes  and  place  increasing 
amounts  in  a  series  of  test-tubes  as  follows:   0.01,  0.05,  0.08,  0.1,  and  0.2  c.c.;    add 
1  c.c.  of  fresh  guinea-pig  complement  serum  1 : 20,  and  1  c.c.  of  2^  per  cent,  suspension 
of  washed  dog  erythrocytes.     Incubate  for  two  hours. 

(a)  Describe  the  changes  occurring  in  the  liver  and  kidneys.     Give 
the  prevailing  views  regarding  the  mechanism  of  their  production. 

(b)  Are  there  any  evidences  of  a  hemolytic  action  of  the  serum  in 
vivof    In  vitro? 

(c)  How  do  you  explain  the  presence  o.f  hemolysin  in  this  immune 
serum? 

(d)  Discuss  the  specificity  of  cytotoxins  in  general. 

(e)  How  may  a  hemolysin  be  removed  from  a  nephrocytotoxic 
serum? 

(f)  Have  the  cytotoxins  any  practical  value  in  the  treatment  of  dis- 
ease? 


EXERCISE  53.— MIOSTAGMIN  REACTION 
EXPERIMENT  106. — TECHNIC  OF  THE  MIOSTAGMIN  REACTION 

1.  Secure  a  portion  of  dry  pulverized  cancer  tissue  and  prepare  an  antigen  after 
the  technic  described  in  the  text. 

2.  Titrate  this  antigen. 

3.  Secure  four  specimens  of  serum:    one  from  a  known  case  of  cancer;   one  from 
a  normal  person,  and  two  for  diagnosis. 

4.  Proceed  with  the  reaction  as  described  in  the  text. 

(a)  Discuss  the  principles  of  this  reaction. 

(b)  Discuss  the  practical  value  of  this  reaction  in  the  diagnosis  of 
cancer. 


EXERCISE  54.— ANAPHYLAXIS 

For  experiments  in  anaphylaxis  sensitize  a  series  of  animals  as  follows: 
(a)  Give  seven  young  guinea-pigs  an  intraperitoneal  injection  of  0.01 
c.c.  horse  serum  (1  c.c.  of  a  1  : 100  dilution). 


ANAPHYLAXIS  941 

(b)  Give  three  young  guinea-pigs  an  intraperitoneal  injection  of  0.01 
c.c.  of  human  serum. 

(c)  Give  two  young  guinea-pigs  an  intraperitoneal  injection  of  0.1 
c.c.  egg-albumen  (1  c.c.  of  1  : 10  dilution). 

(d)  Give  two  rabbits  1  c.c.  of  horse  serum  intravenously  every  three 
days  for  three  doses. 

(e)  Give  a  dog  10  c.c.  of  horse  serum  subcutaneously. 

EXPERIMENT    107. — ANAPHYLAXIS  IN  THE   GUINEA-PIG.     SPECIFICITY 

OF  ANAPHYLAXIS 
1.  Two  weeks  after  the  sensitizing  injections  proceed  as  follows: 

(a)  Inject  a  guinea-pig  sensitized  to  horse  serum  with  1  c.c.  horse  serum  intra- 
peritoneally. 

(b)  Inject  a  guinea-pig  sensitized  to  human  serum  with  0.1  c.c.  of  human  serum 
intravenously. 

(c)  Inject  a  guinea-pig  sensitized  to  egg-albumen  with  1  c.c.  of  diluted  (1 :  4) 
albumin  intraperitoneally. 

(d)  Inject  a  guinea-pig  sensitized  to  horse  serum  with  1  c.c.  of  diluted  (1:4)  egg- 
albumen. 

(e)  Inject  a  guinea-pig  sensitized  to  human  serum  with  0.1  c.c.  horse  serum 
intravenously. 

(f)  Inject  a  guinea-pig  sensitized  to  horse  serum  with  1  c.c.  human  serum  intra- 
peritoneally. 

2.  Carefully  study  the  symptoms  of  anaphylaxis.  After  death  autopsy  the 
animals. 

(a)  Describe  acute  anaphylactic  shock  in  the  guinea-pig.     Does 
death  always  occur? 

(b)  How  soon  after  the  intraperitoneal  injection  of  the  intoxicating 
dose  do  symptoms  develop?     After  the  intravenous?     Why  the  differ- 
ence? 

/  (c)  Is  anaphylaxis  a  specific  reaction? 
.,^7   (d)  What  are  anaphylactogens? 

-/      (e)  Discuss  the  prevailing  views  regarding  the  mechanism  of  the 
anaphylactic  reaction. 

(f)  Describe  the  anatomic  changes  occurring  in  acute  and  fatal 
anaphylaxis  in  the  guinea-pig.  Discuss  the  cause  and  nature  of  these 
changes. 

EXERCISE  55.— ANAPHYLAXIS  (Continued) 
EXPERIMENT  108. — NATURE  OF  THE  ANAPHYLATOXIN 

1.  Place  15  grams  of  coli  bacterial  substance  prepared  as  described  on  page  128 
in  a  distilling  flask  and  add  250  c.c.  of  a  2  per  cent,  caustic  soda  in  absolute  ethyl 
alcohol.  Place  on  a  water-bath  attached  to  a  reflux  condenser  and  collect  the  dis- 
tillate. 


942  EXPERIMENTAL    INFECTION   AND    IMMUNITY 

2.  Evaporate  the  distillate  to  dryness  and  for  each  10  c.c.  of  the  distillate  add 
5  c.c.  of  water.     Neutralize  with  ^  hydrochloric  acid. 

3.  Inject  5  c.c.  intraperitoneally  into  young  guinea-pigs.     Inject  1  or  2  c.c.  intra- 
venously. 

(a)  Do  anaphylactic  symptoms  develop? 

(b)  Are  the  anatomic  changes  in  the  lungs  similar  to  those  observed 
in  serum  anaphylaxis? 

(c)  Discuss  the  prevailing  views  of  the  nature  of  anaphylatoxins. 

(d)  Discuss  the  prevailing  views  of  the  anaphylaxis  antibody,  toxo- 
gen,  or  anaphylactin. 

EXERCISE  56.— ANAPHYLAXIS  (Continued) 
EXPERIMENT  109. — ANAPHYLAXIS  IN  THE  DOG 

1.  Five  weeks  after  sensitization  anesthetize  a  dog  with  ether  and  connect  the 
carotid  or  femoral  artery  with  a  mercurial  manometer  and  arrange  for  a  kymographic 
tracing. 

2.  After  recording  the  normal  blood-pressure  tracing,  inject  5  c.c.  of  homologous 
serum  (horse)  into  a  jugular  vein. 

3.  Record  the  blood-pressure. 

4.  Study  the  coagulation  time  of  the  blood. 

5.  Autopsy. 

(a)  Describe  the  blood-pressure  changes.     To  what  may  they  -be 
ascribed? 

(b)  Is  blood  coagulation  delayed?     Give  a  reason  for  this  change. 

(c)  Do  anatomic  changes  occur  in  anaphylaxis  of  the  dog? 


EXERCISE  57.— ANAPHYLAXIS  (Continued) 
EXPERIMENT  110. — PASSIVE  ANAPHYLAXIS 

1.  Secure  1  c.c.  of  serum  from  a  rabbit  sensitized  to  horse  serum  five  weeks  previ- 
ously and  inject  0.5  c.c.  intraperitoneally  into  each  of  two  guinea-pigs. 

2.  Twenty-four  and  forty-eight  hours  later  inject  both  animals  intravenously 
with  0.2  c.c.  of  horse  serum. 

(a)  Are  anaphylactic  symptoms  in  evidence? 

(b)  Discuss  the  nature  of  passive  anaphylaxis. 

(c)  If  allowed  to  live,  would  these  animals  become  sensitized  to  rab- 
bit serum? 


EXERCISE  58.— ANAPHYLAXIS  (Continued) 
EXPERIMENT  111.— ANTI-ANAPHYLAXIS 

1.  Eight  days  after  sensitizing  a  guinea-pig  with  horse  serum,  inject  1  c.c.  of 
horse  serum  subcutaneously. 


ANAPHYLAXIS  943 

2.  Fifteen  days  after  sensitizing  a  guinea-pig  with  horse  serum,  inject  2  c.c.  of 
serum  into  the  rectum. 

3.  Fifteen  days  after  sensitizing  a  guinea-pig  with  horse  serum,  inject  0.0001  c.c. 
serum  subcutaneously  every  fifteen  minutes  for  six  doses. 

4.  On  the  sixteenth  day  after  sensitizing,  inject  a  guinea-pig  with  1  c.c.  of  horse 
serum.     If  this  animal  develops  anaphylactic  shock,  inject  the  other  three  guinea- 
pigs  in  a  similar  manner. 

(a)  Do  anaphylactic  symptoms  develop?     If  not,  why  not? 

(b)  Discuss  the  importance  of  quantitative  factors  in  producing 
anti-anaphy  laxis . 

(c)  Discuss  the  importance  of  anaphylaxis  and  anti-anaphylaxis 
in  serum  therapy. 

(d)  What  method  is  generally  employed  in  the  effort  to  induce  an 
anti-anaphylactic  state  in  serum  therapy? 

(e)  What  drug  has  been  found  experimentally  of  value  in  ameliorat- 
ing or  preventing  the  pulmonary  changes  of  anaphylaxis? 

(f)  Do  anesthetics  prevent  anaphylaxis? 

(g)  Under  what  conditions  would  you  expect  anaphylaxis  in  persons? 


EXERCISE  59.— ANAPHYLAXIS  (Continued) 
EXPERIMENT  112. — LOCAL  ANAPHYLACTIC  REACTIONS 

1.  Shave  the  abdomen  of  the  rabbit  sensitized  to  horse  serum  five  weeks  after 
the  time  of  sensitization. 

2.  Make  two  superficial  abrasions  over  the  upper  portion  of  the  abdomen;  into 
one  rub  horse  serum,  and  in  the  other,  normal  salt  solution. 

(a)  Does  a  local  reaction  occur?     If  so,  describe  the  lesion. 

3.  Then  inject  0.01  c.c.  of  serum  subcutaneously.     If  acute  anaphylactic  death 
does  not  follow,  observe  the  animal  during  the  next  twenty-four  hours. 

(a)  Does  a  local  Arthus  reaction  follow  this  injection  of  serum?    If 
so,  describe  the  reaction. 

(b)  Explain  the  mechanism  of  a  local  anaphylactic  reaction. 

(c)  Are  the  tuberculin,  luetin,  and  mallein  reactions  anaphylactic? 
Explain  their  mechanism  and  discuss  their  practical  value  in  diagnosis. 

4.  Inject  1  c.c.  horse  serum  .intravenously.     If  anaphylaxis  occurs,  record  the 
symptoms.     Autopsy  with  particular  attention  to  the  heart. 


944  EXPERIMENTAL   INFECTION   AND    IMMUNITY 

EXERCISE  60.-CHEMOTHERAPY 
EXPERIMENT  113. — SALVARSAN 

1.  Secure  a  white  rat  infected  with  Sp.  recurrens  (Sp.  Obennayer)  and  one  withT. 
equiperdum.     Also  two  normal  rats. 

2.  Examine  a  drop  of  blood  from  the  tail  of  each  infected  rat  and  form  a  rough 
estimate  of  the  number  of  parasites  per  microscopic  field. 

3.  Inject  both  rats  intravenously  with  0.001  gm.  salvarsan  freshly  prepared. 

4.  Examine  the  blood  at  the  end  of  two  hours;   after  forty-eight,  seventy-two, 
and  ninety-six  hours. 

5.  Inject  a  control  rat  intravenously  with  0.01  gm.  salvarsan  and  the  second  with 
0.05  gm. 

(a)  Do  the  control  rats  survive? 

(b)  Are  the  infected  rats  sterilized? 

(c)  What  is  meant  by  the  dosis  lethalis?    By  the  dosis  curativa 
By  the  dosis  tolerataf 

(d)  Discuss  organotropism  and  parasitotropism. 

(e)  Discuss  "drug  fastness." 

(f)  Discuss  the  question  of  chemoreceptors. 


INDEX 


ABDERHALDEN'S  reaction,  Bronfenbren- 

ner's  modification,  273 
mechanism,  260 
serodiagnosis  of  pregnancy,  265 
dialyzation  method,  265,  266 
blood-serum,  271 
glassware,  266 
ninhydrin,  267 
precautions,  266 
preparation  of  placental  tis- 
sue, 270 

reading  reaction,  273 
reagents,  267' 
sources  of  error,  274 
testing  dialyzing  shell,  266 
shell  for  non-permeability 

to  albumin  268 
for  permeability  to  pep- 
tone, 269 

experimental  work,  915 
optical  method,  265,  274 
placental  peptone,  274 
polariscope,  276 
reading  reaction,  276 
testing  peptone,  275 
practical  value,  276 
Abortin  reaction,  648 
Abortion,  contagious,  complement-fixa- 
tion test  in,  513 
Abrin,  226 

toxin,  120 

Abscesses,  vaccine  therapy,  704 
Absorption  agglutination  reaction,   ex- 
perimental work,  919 
in  mixed  infection,  309 
by  colloids,  547 
methods  for   differentiating  between 

mixed  and  single  infection,  285 
Abwehrfermente,  259 
Acanthosis  nigricans,  salvarsan  in,  881 
Acetone-insoluble    lipoids    for    Wasser- 

mann  reaction,  446 
Acid  agglutination,  283 

test,  technic,  310 
Acid-fast    bacilli,    fixing    and    staining, 

200 
Acids  and  alkalis,  hemolytic  influence  of, 

experimental  works,  924 
Acne,  vaccine  therapy,  704 
Actinocongestin,  569 
Adenitis,  tuberculous,  tuberculin  treat- 
ment, 731 


Adulteration,   meat,   detection  of,   bio- 
logic blood  test  for,  333 
technic,  335 
Agglutinating  power  of  serum,  variation, 

288 

serums,  68 
Agglutination,  279 
acid,  283 

group,  experimental  work,  918 
mechanism  of,  284 
Bordet's  theory,  284 
Gruber  theory,  284 
Paltauf  s  theory,  284 
of  treponema  pallidum,  292 
test,  294 

acid,  technic,  310 

absorption,  experimental  work,  919 

in  mixed  infection,  309 
in  cerebrospinal  meningitis,  292 
in  cholera,  292 
in  diagnosis  of  disease,  290 
in  differentiation  of  pneumococci, 

308 

in  dysentery,  292 
in  glanders,  293 
in  identification  of  micro-organism, 

293 

in  Malta  fever,  292 
in  measuring  immunizing  response, 

294 

in  pertussis,  293 
in  plague,  292 
in  pneumonia,  292 
in  single  or  mixed  infection,  294 
in  treponema  pallidum,  292 
in  tuberculosis,  292 
in  typhoid  fever,  290,  295,  299 
in  whooping  cough,  293 
macroscopic,  295 

experimental  work,  917,  918 
Kolle  and  Pfeiffer's  method,  305 
Oxford  University  method,  305 
technic,  301 
microscopic,  295 

dry  method,  technic,  300 
experimental  work,  916 
wet  method,  technic,  299 
practical  applications,  290 
precautions,  298 
preparation  of  emulsion,  296 
requisites  for,  295 
Agglutinins,  154,  279 


60 


945 


946 


INDEX 


Aggultinins,  absorption  methods  for  dif- 
ferentiating between  a  mixed  and 
single  infection,  285 
action  of,  based  on  collodial  reactions, 

550 

definition,  279 
experimental  work,  891,  916 
formation,  281 
group,  285 
history,  279  ' 
immune,  281 
in  immunity,  role  of,  289 
nature,  283 
normal,  281 
origin,  282 
partial,  285 

preservation  of,  in  dried  paper  form,  80 
production  of,  68 

intraperitoneal  method,  69 
intravenous  method,  68 
properties,  283 
specificity,  285 
Agglutinogen,  282 

experimental  work,  919 
Agglutinoids,  281 
Agglutinoscope,  305 
Aggressins,  109,  124,  184 
anti-,  127 
artificial,  104,  126 
in  relation  to  infection,  104 
natural,  126 

experimental  work,  900 
nature  of,  126 
Albuminolysin,  588 

Alcoholic    extracts    of    normal    organs 
for     Wassermann     reaction, 
444 
re-enforced  with  cholesterin  for 

Wassermann  reaction,  445 
of  syphilitic  livers  for  Wassermann 

reaction,  444 

Aleppo  boil,  salvarsan  in,  881 
Aleuronat,  preparation  of,  361 
Alexin.     See  Complement. 
Alkalis  and  acids,  hemolytic  influence, 

experimental  work,  924 
Allergen,  572,  579 
Allergic  skin  reactions,  620.     See  also 

Anaphylactic  reaction. 
Allergin,  588 
Allergy,  570,  572 
Amboceptoids,  343 
Amboceptor,  156, 159, 187,  338,  341,  348, 

359,  360 

and   complement,    quantitative   rela- 
tionship, 396 
anti-,  347 
bacteriolytic,  157,  341 

titration  of,  347 
formation,  344 
hemolytic,  341,  384 

and  complement,   quantitative  re- 
lationship, 396 

for  Noguchi's  modification  of  Was- 
sermann reaction,  476 


Amboceptor,  hemolytic,  for  Wassermann 

reaction,  439 
preservation  of,  80 
titration  of,  347 

experimental  work,  925 
history,  341 
"mechanism  of  action,  343 

experimental  work,  927 
native,  346 
natural,  346 
properties,  343 

experimental  work,  927 
quantitative  estimation,  347 
role    of,    in    hemolysis,    experimental 

work,  926 
specificity,  345 

experimental  work,  926 
structure,  342 
unit  of  serum,  396 
Amebadiastase,  185 
Amorphous  colloids,  544 
Amoss  and  Flexner    method    for    rapid 
production  of  antidysenteric  serum, 
247 

and  Wollstein's  method  for  rapid 
production  of  antimeningococcus 
serum,  792 

Ampules,   vaccine,    conversion   of  test- 
tubes  into,  24 
making  of,  24 

Anaphylactic  antibody,  588 
reactions,  620 

as  indices  of  immunity,  622 

of  infection  and  hypersusceptibil- 

ity,  621 

effects  of  drugs  upon,  644 
etiology,  620 
experimental  work,  943 
in  diphtheria,  652 
in  gonococcus  infection,  652 
in  leprosy,  653 
in  pregnancy,  653 

in  serum  and  food  hypersensitive- 
ness,  652 

in  sporotrichosis,  653 
in  typhoid  fever,  649 
prognostic  value,  653 
Anaphylactin,  588,  592,  601 
nature,  588 
terminology,  588 
Anaphylactogens,  572,  578,  579 
bacterial,  583 
endotoxins  as,  584 
non-protein,  579 
physical  state,  582 
protein,  chemistry,  580 
Anaphylactotoxins,  578 
Anaphylatoxin,  572,  584 

theory  of  anaphylaxis,  584 
Anaphylaxis,  567 

accelerated,  in  infectious  diseases,  605 
anaphylatoxin  theory  of,  584 
antibody,  588,  601 
Arthus  phenomenon,  569 
Besredka's  theory,  591 


INDEX 


947 


Anaphylaxis,  cellular  theory,  594 
chronic,  576 
definition,  571 
desensitization  in,  599 
experimental  work,  940 
fall  in  blood-pressure  in,  577,  578 
food,  615 

Friedberger's  theory,  592 
Gay  and  Southard's  theory,  592 
Hamburger  and  Moro's  theory,  591 
heterologous,  597 
history,  568 
homologous,  597 
humoral  theory,  584,  591 
immediate,  in  infectious  diseases,  605 
in  cats,  575 

in  cold-blooded  animals,  577 
in  cows,  577 
in  dogs,  576 

experimental  work,  942 
in  guinea-pig,  573 

experimental  work,  941 
in  hens,  577 
in  horse,  577 
in  man,  573 
in  pigeons,  577 
in  rabbits,  575 
in  relation  to  immunity,  602 

to  infection,  602 
in  serum  treatment,  738 
in  sheep,  577 
in  white  mice,  576 

rats,  576 
indirect,  579 
lipoid,  580 
mechanism,  577,  598 
nature  of,  experimental  work,  941 
Nolf's  theory,  593 
passive,  584,  596 

production  of,  597,  942 
physical  theory,  593 
protein  matrix  in,  587 
relation  of,  to  infectious  diseases,  603 

to  non-infectious  diseases,  608 
Richet's  studies  on,  569 

theory,  591 

Smith's  phenomenon,  571 
specificity,  601 
terminology,  572 
theories,  591 
torpid    early,   in    infectious    diseases, 

605 

Vaughn  and  Wheeler's  theory,  592 
von  Pirquet's  studies  on,  569 
Anemia,  salvarsan  in,  881 
Anesthesia  for  subdural  inoculation  of 

serum,  749 

in  lumbar  puncture,  39 
Wassermann  reaction  after,  491 
Angina,  Vincent's,  salvarsan  in,  881 
Animal  blood.     See  Blood,  animal. 
cold-blooded,  anaphylaxis  in,  577 
conjunct! val  tuberculin  test  in,  641 
cutaneous  tuberculin  test  in,  642 
experiments,  891 


Animal   immunization,   active,    general 

technic,  66 

methods  for  effecting,  65 
inoculation,  53 
intracardial,  62 

of  guinea-pigs,  62 
intramuscular,  56 
intraperitoneal,  64 
of  guinea-pig,  64 
of  rabbit,  64 
intravenous,  56 
of  dog,  62 
of  goats,  62 
of  guinea-pig,  57 
of  horse,  61 
of  mice,  60 
of  rabbit,  56 
of  rats,  60 
of  sheep,  62 
subcutaneous,  54 

with  fluid  inoculum,  54 
with  solid  inoculum,  55 
technic,  53 

general  rules,  53 
intracutaneous     tuberculin     test    in, 

642 

parasites,  aggressiveness,  135 
infection  with,  134 

mode,  134 

production  of  disease  by,  135 
preparation  of,  for  vaccination,  672 
protective  immunization,  700 
subcutaneous  tuberculin  test  in,  641 
tuberculin  reaction  in,  641 
vaccination  of,  672 

preparation  for,  672 
Anopheles  maculipennis,  168 

punctipennis,  168 
Antenatal  infection,  90 
Anthrax,  internal,  salvarsan  in,  821 

serum  treatment,  820,  821 
vaccination,  700 
Antiaggressins,  127 
Antiamboceptors,  347 
Antianaphylaxis,  599,  739 

experimental  production,  599,  942 
mechanism,  600 
Anti-anthrax  serum,  820 
administration,  820 
Antibacterial  immunity,  736 
acquired,  172 
experimental  work,  902 
immunization,  736,  787 
Antibody,  65,  66,  150,  151,  163,  339 
anaphylactic,  588 
anaphylaxis,  588,  601 
and  antiferments,  similarity  between, 

257 

of  first  order,  161 
of  second  order,  154 
of  third  order,  156 
specificity  of,  164 

tissues  concerned  in  production,  164 
Anticholera  serum,  823 
Kraus',  823 


948 


INDEX 


Anticomplementary    action    of    serums, 

experimental  work,  933 
titration  of  antigens  in  Wassermann 

reaction,  452 
Anticomplements,  349 

auto-,  349 

Anticytotoxic  serums,  538 
Antidiphtheric  serum,  production  of,  235 
Antidysenteric    serum,    collecting    and 

testing,  248 
production  of,  245 
culture,  246 

immunizing  animals,  247 
rapid    method    of    Flexner    and 

Amoss,  247 
Antiferments,  256 

and  antibodies,  similarity  between,  257 
experimental  work,  914 
in  disease,  257 
simple,  151 
Antigen^6M>fir..67,  150,  161 

bacterial,    identification    of,    comple- 
ment-fixation test  for,  531 
preparation  of,  in  complement  fixa- 
tion, 498 
principles    of    complement   fixation 

with,  501 

standardizing,  in  complement  fixa- 
tion, 500 
determination     of,     by     complement 

fixation,  527 
for  antimeningococcus  serum,  Hitchens 

and  Hansen's  method,  793 
for    gonococcus    complement-fixation 

test,  505 

for  Noguchi's  modification  of  Wasser- 
mann reaction,  478 
titration  of,  479 
for  Wassermann  reaction,  441 

anticomplementary  titration,  452 
antigenic  titration,  455 
experimental  work,  932 
hemolytic  titration,  454 
method  of  diluting,  451 

of  titrating,  452 
preparation,  443 
gonococcus,  titration  of,  experimental 

work,  936 
non-protein,  161 

Antigenic  dose  of  syphilitic  serum,  442 
titration  in  Wassermann  reaction,  455 
values  of  various  extracts  in  serum 

diagnosis  of  syphilis,  448 
Antigonococcus  serum,  818 
-  action,  819 
administration,  819 
preparation,  819 
Antihemolysins,  394 
Anti-influenza  serum,  ,803 

administration,  804 
Antilactase,  256 
Antilysin    test    for    antistaphylococcus 

serum,  249 

Antimeningococcus  serum,  788 
action,  794 


Antimeningococcus    serum,  administra- 
tion, 795 

Amoss  and  Wollstein's  rapid  method 
of  production,  792 

antigen  for,  Hitchens  and  Hansen's 
method  of  preparing,  793 

dosage,  795 
repeating,  797 

Flexner   and    Jobling's    method    of 
preparing,  791 

intravenous  injection,  796 

preparation,  791  ^ 

repeating  doses,  797 

serum  sickness  from,  799 

standardization,  792 

subdural  inoculation,  795 
Antipepsin,  256 
Antiplague  serum,  822 
Antipneumococcus  serum1,  810 

action,  811 

administration,  812 

concentration,  811 

intravenous  injection,  812 

Kolle's  method  of  preparing,  811 

preparation,  810 

results  from,  812 

standardization,  811 
Antiprecipitin,  318 
Antirennin,  256 
Antisensibilisin,  592 

Antiseptics,  preservation  of  serum  by,  76 
Antistaphylococcus  serum,  249,  819 

antilysin  test  for,  249 

preparation,  249 
Antistaphylolysin,    experimental    work, 

913 

method  of  titrating,  in  serum,  251 
Antisteapsin,  256 
Antistreptococcus  serum,  813 

action,  814 

administration,  816 

in  bronchopneumonia,  817 

in  diphtheria,  817 

in  endocarditis,  817 

in  erysipelas,  817 

in  puerperal  sepsis,  817 

in  scarlet  fever,  817 

in  smallpox,  817 

in  tuberculosis,  817 

in  wound  infections,  817 

intravenous  injection,  816 

Kolle's  method  of  preparing,  816 

preparation,  815 

standardization,  816 

value,  817 

Antitetanic  serum,  production  of,  243 
Antitetanolysin   action  of,  experimental 

work,  912 
Antitoxic  immunity,  73? 

acquired,  173 
passive,  175 
immunization,  736,  754 
Antitoxin,  151,  224 
action  of,  based  on  colloidal  reactions, 

548 


INDEX 


949 


Antitoxin,  action  of,  on  toxin,  232 
antidysenteric,  245 
antistaphylococcus,  249 
botulinus,  245 
definition,  224 
diphtheria,  754.     See  also  Diphtheria 

antitoxin. 
dysentery,  782 

administration  and  uses,  782 
experimental  work,  911 
formation,  225 
hay-fever,  252,  786 
history,  224 
immunity,  natural,  171 
measure  of,  252 
method  of  concentrating,  239 
natural,  228 

diphtheria,  Schick  test,  288 
pollen,  252,  786 
practical  application,  253 
production  of,  68 
by  horses,  238 

for  therapeutic  purposes,  234 
properties,  227 
relation  of,  to  proteids,  227 
specificity,  232 

experimental  work,  912 
structure,  227 

tetanus,  772.     See  also  Tetanus  anti- 
toxin. 
unit,  252 

Behring-Ehrlich,  240 
Antitrypsin,  256 
test,  264 

Bergmann  and  Meyer's,  264 
Antituberculosis  serum,  824 
Antityphoid  serum,  821 
Antityrosinase,  256 
Antiurease,  256 
Antivenene,  Calmette's,  786 
Antivenin,  preparation  of,  252 

production  of,  251 
Apotoxin,  591 
Aqueous  extract  of  pallidum  culture  for 

Wassermann  reaction,  448 
of  syphilitic  livers  for  Wassermann 

reaction,  443 
Arsenoxid,  878 

Arthritis,  gonorrheal,  autoserum  treat- 
ment, 836 

vaccine  therapy,  707 
Arthus  phenomenon  of  anaphylaxis,  569 
Artificial  aggressins,  104,  126 
Ascoli  and  Izar's  miostagmin  test,  563 

See  also  Miostagmin  reaction. 
Asthma,  horse,  615 

in  serum  treatment,  739 
Athrepsia,  172 
Athreptic  immunity,  172 
Atmosphere,  bacteria  in,  85 
Atrophy,  receptoric,  80,  173 
Atropin  sulphate  as  preventive  of  serum 

disease,  613 

Auto-anticomplements,  349 
Autocytotoxins,  537 


Autogenous  bacterial  vaccines,  665 
Autolysins,  385 
Autonephrotoxins,  537 
Autopsies,  891 
Autoserum  treatment,  835 

mechanism   of   therapeutic   action, 

840 

of  acute  infectious  diseases,  835 
of  cancer,  843 
of  chorea,  836 
of  erysipelas,  835 
of  gonorrheal  arthritis,  836 
of  hydrocele,  842 
of  influenza,  836 
of  leprosy,  835 
of  Malta  fever,  836 
of  marasmus,  843 
of  non-tuberculous  effusions,  843 
of  pellagra,  836 
of  pneumonia,  836 
of  skin  diseases,  835 
of  tuberculosis  of  serous  membranes. 

841 
of  tuberculous  meningitis,  843 

pleurisy,  841 
of  typhoid  fever,  836 
salvarsanized,  in  syphilis  of  brain, 

836 

after-treatment,  839 
repeating  dose,  840 
serobiologic  findings  in  cere- 

brospinal  fluid,  840 
technic,  838 
Autumnal  catarrh,  252 
Ayer  and  Gay's  method  of  titration  of 
bacteriolytic  complement,  356 


BACILLEN  emulsion,  tuberculin,  prepara- 
tion, 718 
Bacillus  abortus,  648,  649 

acid-fast,  fixing  and  staining,  200 

botulinus,  117 

diphtheria,  113 

virulence  and  toxicity,  method  of 
testing,  894 

dysentery,  118 

tetanus,  117 

tubercle,  living,  in  treatment  of  tuber- 
culosis, 721 
Bacteremia,  93 
Bacteria,  83 

agglutination  reaction  in  identification 
of,  293 

aggressiveness  of,  95 

Bail's  classification,  125 

conglutination  test  with,  307 

defensive  mechanism  of,   in  relation 
to  infection,  103 

effect  of  opsonins  on,  192 

fixing  and  staining,  200 

in  atmosphere,  85 

in  foods,  85 

in  milk,  85 

in  placenta,  86,  90 


950 


INDEX 


Bacteria  in  water,  85 

mechanical  action,  109,  133 

experimental  action,  902 
morphologic   changes,   in  relation  to 

infection,  103 

non-agglutinable  species,  288 
non-pathogenic,  84 
numeric  relationship,  to  infection,  99 
on  skin,  85,  87 
pathogenic,  83,  84 
physiologic    changes,    in    relation    to 

infection,  103 

recovered  from  feces,  water-supplies, 
etc.,  bacteriolytic  test  for  identifica- 
tion, 365 

selective  affinity,  99 
toxicity  of,  95,  96 

transmission  of,  by  suctorial  insects,  86 
virulence  of,  95 
decrease,  96 
increase,  96 

by  addition  of  animal  fluids  to 

culture-medium,  97 
by  passage  through  animals,    96 
by  use  of  collodion  sacs,  97 
Bacteria-free  nitration,  preservation  of 

serum  by,  76 
Bacterial  anaphylactogens,  583 

antigens,    identification    of,    comple- 
ment-fixation test  for,  531 
preparation,    in   complement   fixa- 
tion, 498 
principles  of   complement   fixation 

with,  501 

standardizing,  for  complement  fixa- 
tion, 500 
chemotherapy,    bactericidal    tests   in, 

883 
diseases,  chemotherapy  in,  881 

complement  fixation  in  498 
emulsion  in  opsonic  index,  197 
ferments,  254 

invasion,  factors  preventing,  90 
mechanism,  91 
normal  defenses  against,  90 
precipitinogens,  preparation,  336 
precipitins,  experimental  work,  922 
precipitin  test  with,  320 

technic,  336 
production  of,  71 
proteins,  109,  127 
action,  129 

experimental  work,  900 
nature,  128 
Vaughan's  theory,  130 
split  proteins,  128 
toxins,  109.     See  also  Toxins. 
vaccines,  210,  656.     See  also  Vaccines, 

bacterial. 

Bactericidal  power  of  blood,   capillary 

pipet  method  of  measuring,  377 

Neisser  and  Wechsberg's  method 

of  measuring,  371 
plate  culture  method  for  measur- 
ing, 371 


Bactericidal  power  of  blood,  plate  cul- 
ture method,  technic,  372 
Stern  and  Korte's  experimental 

work,  939 

method  of  measuring,  371 
Topfer    and    Jaffe's    method    of 

measuring,  374 
Wright's  capillary  pipet  method 

of  measuring,  377 
Bactericidins,  359 
Bacterin,  656 

therapy,  654,  657,  660 

history,  654 
Bacteriolysins,  156,  340,  358 

and    hemolysins,    analogy    between, 

389 

definition,  359 
experimental  work,  891 
history,  358 

influence  of,  on  endotoxins,  362 
normal,  363 
origin,  360 

practical  applications,  363 
production  of,  69 
properties,  363 
specificity,  363 
Bacteriolysis,  148,  358 

and  hemolysis,  analogy  between,  389 
experimental  work,  939 
mechanism,  362 
Bacteriolytic  amboceptor,  157,  341 

titration  of,  347 
complement,  titration  of,  356 

Gay  and  Ayer's  method,  356 
power  of  blood,  microscopic  method  of 

measuring,  939 

serum  in  treatment  of  disease,  381 
method  of  titrating,  366 
production  of,  69 

test  for  identification  of  bacteria  re- 
covered from  feces,  water-sup- 
plies, etc.,  365 

method  of  testing  virulence  of  cul- 
ture, 365 
of  titrating  bacteriolytic  serum, 

366 
Pfeiffer's,  364 

experimental  work,  938 
in  diagnosis  of  disease,  370 
technic  of,  369 

preparing  immune  serum,  365 
technic,  364 

Bacteriotropins,  147,  187,  191 
experimental  work,  908 
quantitative  estimation,  experimental 

work,  910 

Neufeld's  technic,  203.  See  slso 
Neufeld's  quantitative  estima- 
tion. 

Simon's  method,  206 
Bail's  classification  of  bacteria,  125 

hypothesis,  124 

Bandi  and  Terni's  plague  vaccine,  696 
Bang's   disease    among    cattle,    abortin 
test,  648 


INDEX 


951 


Bauer's  modification  of  Wassermann  re- 
action, 482 

B.  E.  tuberculin,  preparation  of,  718 
Behring-Ehrlich  antitoxin  unit,  240 
Behring's     method     of     immunization 

against  diphtheria,  769 
Beraneck's  tuberculin,  dose  of,  727* 

preparation  of,  718 
Bergmann  and  Meyer's  antitrypsin  test, 

264 
Berkefeld  filter,  77 

cleaning  and  care  of,  78 
Besredka's  theory  of  anaphylaxis,  591 
B.  F.  tuberculin,  preparation  of,  718 
Biologic  blood  test  for  detection  of  blood- 
stains, 326 
technic,  331 
of  meat  adulteration,  333 

technic,  335 
Blackfan's  apparatus  for  collecting  blood, 

36 

Blackleg  vaccination,  702 
Blood,  animal  and  human,  differentia- 
tion, precipitin  test  for,  326 
obtaining  large  amounts,  from  dog, 

51 

from  guinea-pig,  46,  47 
from  hog,  50 
from  horse,  51,  52 
from  monkey,  50 
from  rabbit,  42-47 

Nuttall's  method,  43 
from  rats,  47 
from  sheep,  48 
small  amounts,  41 
from  dog,  51 
from  guinea-pig,  41 
from  horse,  51,  52 
from  monkey,  50 
from  rabbit,  41 
from  rats,  47 
from  sheep,  42,  49 
bactericidal    power,    capillary    pipet 

method  of  measuring,  377 
Neisser  and  Wechsberg's  method 

.of  measuring,  371 
plate  culture  method  for  measur- 
ing, 371 
technic,  372 
Stern  and  Korte's  experimental 

work,  939 

method  of  measuring,  371 
Topfer    and    Jaffe's    method    of 

measuring,  374 
Wright's  capillary  pipet  method 

of  measuring,  377 

bacteriolytic  power,  microscopic 
method  of  measuring,  experimental 
work,  939 

Blackfan's  apparatus  for  collecting,  36 
capsules,  Wright's,  making  of,  23 
method  of  sealing,  34 
removing  serum  from,  34 
collecting  of,   for  Wassermann  reac- 
tion, 432 


Blood,  collecting  of,  for  Wassermann  re- 
action, New  York  Board  of  Health 
outfit,  434 
films  for  phagocytic  counts,  202 

method  of  preparing,  201 
human    and    animal,    differentiation, 

precipitin  test  for,  326 
obtaining  large  amounts,  33 
phlebotomy,  33 
wet  cupping,  36 
small  amounts,  32 

from    infants    and    children, 

33 

Keidel  tube  for  collecting,  35,  36 
methods  of  obtaining,  28 
obtaining,  28 

placenta!,  method  of  obtaining,  37 
plasma,  obtaining,  30 
precipitin  test  for,  326 

technic,  331 

test,  biologic,  for  detection  of  blood- 
stains, 326 
technic,  331 
of  meat  adulteraton,  333 

technic,  335 
Teichmann's,  326 
transfusion,  287,  830 
experimental  work,  920 
methods,  831 

relation  of  isohemagglutinins,  286 
technic,  832 
tests  before,  for  isohemagglutinins 

and  isohemolysins,  311 
Brem's  microscopic  hemag- 
glutination  methods,  312 
Blood-corpuscles  and  blood-serum,  ob- 
taining, 30 

red.     See  Erythrocytes. 
Blood-pressure  as  guide  in  administering 

serum  subdurally,  747 
fall  in,  in  anaphylaxis,  577,  578 
Blood-serum.     See  Serum. 
Blood-stains,  biologic  test  for,  326 

technic,  331 

experimental  work,  921,  937 
identification  of,  complement-fixation 

test  for,  527 
Body-fluids,  relation  of,  to  phagocytosis, 

186 

Boil,  Aleppo,  salvarsan  in,  881 
Bones,  tuberculosis  of,  tuberculin  test  in 

diagnosis  of,  631 
treatment,  731 

Bordet-Gengou    phenomenon    of    com- 
plement fixation,  354,  413 
as  colloidal  reaction,  552 
determination   of   antigen    by, 

527 

experimental  work,  931 
for   identification   of   bacterial 

antigens,  531 
of  blood-stains,  527 
of  meats,  531 
in  bacterial  diseases,  498 
in  cancer,  532 


952 


INDEX 


Bordet-Gengou    phenomenon    of    com- 
plement fixation    in    conta- 
gious abortion,  513 
in  dourine,  515 
in  echinococcus  disease,  524 
in  glanders,  511 
in  gonococcus  infections,   503. 
See  also  Gonococcus  comple- 
ment-fixation test. 
in  horse  syphilis,  515 
in  standardization  of  immune 

serums,  524 
in  tuberculosis,  517 
in  typhoid  fever,  516 
mechanism,  417 
non-specific,  418 
original,  416 

practical  application,  423 
preparation  of  bacterial  anti- 
gens, 498 
principles,  413 

with  bacterial  antigens,  501 
protein  differentiation  by,  527 
quantitative  factors  in,  421 
standardizing    bacterial    anti- 
gens, 500 
technic,  425,  498 

Bordet's  theory  of  mechanism  of  agglu- 
tination, 284 

Botulinus  antitoxins,  245 
Botulism  toxin,  117 

experimental  work,  896 
Bouillon  filtrate,  preparation  of,  .718 
Brain,   syphilis  of,   salvarsanized  auto- 
serum  in  836 
after-treatment,  839 
repeating  dose,  840 
serobiologic    findings    in    cere- 

brospinal  fluid,  '840 
technic,  838 
Brem's    microscopic    hemagglutination 

methods,  312 
Brezovsky  and  Detre's  modification  of 

Wassermann  reaction,  484 
Bronchitis,  vaccine  therapy,  710 
Bronchopneumonia,  antistreptococcus  se- 
rum in,  817 
Bronfenbrenner's  modification  of  Abder- 

halden  reaction,  273 
Browning  and  Mackenzie's  modification 

of  Wassermann  reaction,  484 
test  for  estimating  amount  of  com- 
plement absorbed  in  Wassermann 
reaction,  458 
Bubonic  plague,  serum  treatment,  822 

vaccination,  694 
Buckwheat  idiosyncrasy,  615 
Butyric-acid  test,  Noguchi's,  for  protein, 
322 


CADAVER   serums,    testing,    in   Wasser- 
mann reaction,  435 
Calf  cholera  serum,  785 
Calmette's  antivenene,  786 


Calmette's  conjunctival  tuberculin  test. 

628,  639 
dangers,  633 
in  animals,  641 

Cancer,  autoserum  treatment,  843 
chemotherapy  in,  887 
complement-fixation  test  in,  532 
contraindications  to  vaccines  in,  668 
eosin-selenium  compound  in,  888 
epiphanin  reaction  in,  563 
Freund  and  Kaminer's  cytolytic  test 

for,  541 

miostagmin  reaction  in,  565,  566 
sero-enzymes  in,  277 
venom  hemolysis  in,  412 
Capillary  pipet  for  counting  bacterial 

vaccine,  214 

for  opsonic  index  determination,  199 
making  of,  18 
method  of  sealing,  201 
rubber  teats  for,  20 

Capsules,  blood,  Wright's  making  of,  23 
method  of  sealing,  34 
removing  serum  from,  34 
Carbuncle,  antistaphylococcus  serum  in, 

819 

vaccine  therapy,  704 
Castellani's  agglutination  reaction,  ex- 
perimental work,  919 
Cataphoresis,  545 
Catarrh,  autumnal,  252 
Cats,  anaphylaxis  in,  574 
Cattle,  Bang's  disease  among,  abortin 

test,  648 
Cells,  functions  of,  149 

heart  failure,  180 
Cellular  theory  of  anaphylaxis,  594 

of  immunity,  145 
Centrifuge,  17 
care  of,  17 
electric,  18 
Cerebral  syphilis,  Wassermann  reaction 

in,  488 

Cerebrospinal  fluid  for  Wassermann  re- 
action, 435 

method  of  securing,  37 
meningitis,  agglutination  test  in,  292 
precipitin  test  in  diagnosis,  320 
serum  treatment,  788 
vaccination,  699 
syphilis,  Lange's  collidal  gold  reaction 

in,  559 

Chancroid,  salvarsan  in,  881 
Chantemesse's  serum,  821 
Chemoreceptors,  847 
Chemotaxis,  181 

experimental  work,  906 
negative,  147,  181,  184 

experimental  work,  906 
positive,  147,  181 

experimental  work,  906 
Chemotherapeutic  research,  leads  in,  882 
Chemotherapy,  844 

bacterial,  bactericidal  tests,  883 
experimental  work,  944 


INDEX 


953 


Chemotherapy  in  bacterial  diseases,  881 
in  cancer,  887 
in  malignant  disease,  887 
in  pneumococcus  infections,  885 
in  tuberculosis,  887 
principles,  845 

Chills  and  fever  after  intravenous  in- 
jection of  salyarsan,  867 
Cholera,  agglutination  test  in,  292 
hog,  serum  treatment,  784 
serum,  calf,  785 

hog,  production  of,  784 

standardization  of,  784 
treatment,  823 
vaccination,  697 

dosage  of  vaccine,  698 
Haffkine's  vaccine,  697 
Kolle's  vaccine,  697 
preparation  of  vaccine,  697 
results,  698 
Strong's  vaccine,  698 
Cholesterin  and  lecithin  for  Wassermann 

reaction,  447 
Chorea,  autoserum  treatment  in,  836 

salvarsan  in  881 
Claypool  and  Gay's  sentitized  typhoid 

vaccine,  691 
typhoidin,  650 
Cobra  hemotoxin,  122 
lecithid,  352 

venom,  experimental  work,  899 
preparation,  407 
tests,  405.     See  also  Venom  hemoly- 

sis. 

Cobralecithinase,  406 
Cold-blood  animals,  anaphylaxis  in,  577 
Cole's  method  of  determining  type  of 

Sneumococcus,  308 
lapse  in  subdural  inoculation  of  se- 
rum, 746,  748 
symptoms,  748 
treatment,  748 
Colics'  law,  Wassermann  reaction  and, 

489 
Collodion  sacs  for  increasing  virulence  of 

bacteria,  97 
Colloid,  irreversible,  544 

reversible,  544 
Colloidal  reactions,  action  of  agglutinins 

based  on,  550 
of  antitoxins  based  on,  549 
of  hemolysins  based  on,  551 
of  precipitins  based  on,  550 
complement-fixation  test  as,  552 
gold  test  of  Lange,  555 
immunity   reactions   and,    analogy 

between,  548 
Wassermann  test  as,  552 
solutions,  544 
suspensions,  544 
Colloids,  absorption  by,  547 
amorphous,  544 
nature,  543 
osmotic  pressure,  545 
physical  structure,  545 


Colloids,  precipitation,  545,  546,  547 
properties,  543 
relation  of,  to  immunity,  543 
surface  tension,  545 
varieties,  543 

Comer's  automatic  pipet,  219 
Complement,  148, 156, 157, 159, 187,  338, 

341,  348,  359,  384 
action,  351 

and  amboceptor,     quantitative    rela- 
tionship, 396 
bacteriolytic,  titration  of,  356 

Gay  and  Ayer's  method,  356 
definition,  348 
deflection  of,  355 
deviation  of,  355 
dominant,  343,  388 
endocellular,  352 

fixation,  Bordet-Gengou  phenomenon, 
354,  413.     See  also  Bordet-Gengou 
phenomenon. 
non-specific,  by  normal  rabbit,  dog, 

and  mule  sera,  419 
reactions,  experimental  work,  931 
for  identification  of  bacterial  an- 
tigens, 531 
in  cancer,  532 
in  contagious  abortion,  513 
in  differentiation  of  proteins,  ex- 
perimental work,  937 
of  microparasites,  502 
in  dourine,  515 
in  echinococcus  disease,  524 
in  glanders,  511 
in  gonococcus  infections,  503 
in  pertussis,  521 

in  standardization  of  immune  se- 
rums, 524 
in  tuberculosis,  517 
in  typhoid  fever,  516 
non-specific,  418 
practical  applications,  423 
principles  of,  413 
quantitative  factors  in,  421 
technic,  425 

for  Noguchi's  modification  of  Wasser- 
mann reaction,  476 
for  Wassermann  reaction,  435 

preservation,  437 
hemolytic,  experimental  work,  929 
titration  of,  356 

experimental  work,  931 
history,  348 
inactivation  of,    experimental    work, 

936 

multiplicity,  350 
nature,  351 
non-dominant,  343 
origin,  350 
properties,  348 

experimental  work,  930 
reactivation  of,  experimental  work,  930 
r61e   of,    in   hemolysis,    experimental 

work,  926 
splitting,  353 


954 


INDEX 


Complement  structure,  348 

test  as  colloidal  reaction,  552 
titration  of,  quantitative,  356 
Complementoid,  349 
Congenital  mental  deficiency,   Wasser- 

mann  reaction  in,  490 
syphilis,  Wassermann  reaction  in,  489, 

490 

Congestion,  591 
Conglutination,  289 

test  with  bacteria,  technic,  307 
Conglutinin,  289 
Conjunctival    tuberculin    test    of    Cal- 

mette,  628,  639 
dangers,  633 
in  animals,  641 
of  Wolff-Eisner,  628,  639 
dangers,  633 
in  animals,  641 

Contagious  abortion,   complement-fixa- 
tion test  in,  513 
diseases,  84 
Contamination,  81 
Cow-disease,  656 
Cowpox,  656 

vaccine,  preparation,  670 

Noguchi's  method,  674 
animals,  671 

collection  of  virus,  672 
seed  virus,  671 
testing  virus,  674 
Cows,  anaphylaxis  in,  577 
Craig's  modification  of  Wassermann  re- 
action, 482 
Crotin  toxin,  120 
Cryptogenic  infections,  92 
Crystalloids,  543 
Culex,  168 
Cupping,  wet,  36 

Cutaneous  reaction  in  typhoid  fever,  ,650 
tuberculin  reaction   of   von   Pirquet, 

628,  637 
in  animals,  642 
Cyst,  echinococcus,  complement-fixation 

test  in,  524 

Cystitis,  vaccine  therapy,  706 
Cytase,  146,  159,  341,  359,  360 
Cytolysins,  338,  534 
definition,  339 
nomenclature,  340 
varieties,  340 
Cytotoxic  reactions,  541 
in  diagnosis,  541 
of  Freund  and  Kaminer  for  cancer, 

541 

Cytotoxins,  156,  340,  534 
experimental  production,  892 
in  diagnosis,  541 
in  immunity,  role  of,  540 
in  treatment,  540 
methods  of  studying,  535 
nature,  534 
nomenclature,  534 
practical  applications,  540 
preparation,  535 


Cytotoxins,  production  of,  73 

Pearce's  method,  73 
properties,  534 
specificity,  536 
varieties,  538 


DEMENTIA,   paralytic,   Wassermann  re- 
action in,  487 
Dermatitis  herpetiformis,  salvarsan  in, 

881 
venenata,  120 

vaccine  therapy,  705 
Desensitization  in  anaphylaxis,  599 
Detre  and  Brezovsky's  modification  of 

Wassermann  reaction,  484 
Deutero  toxin,  116 
Deviation  of  complement,  355 
Diabetes,  contraindications  to  vaccines 

in,  668 
Diarrhea  after  intravenous  injection  of 

salvarsan,  867 

Diet  as  predisposing  to  infection,  102 
Digestive  tract  as  portal  of  entrance  for 

bacteria,  88 

Diphtheria,  allergic  reaction  in,  652 
antistreptococcus  serum  in,  817 
antitoxin,  754 
action,  757 
administration,  758 
dosage,  761 

in  diphtheria  of  eye,  763 
of  vulva,  763 
of  wounds,  763 
in  nasal  diphtheria,  762 
in  tonsillar  diphtheria,  762 
influence  of  age,  763 
repeating,  763 
total,  764 

early  use,  importance,  759 
intramuscular  injection,  759 
intravenous  injection,  759 
mortality  after  use,  766 

before  use,  765 
natural,  Schick  test,  228 
oral  method  of  administering,  759 
preparation,  755 
production  of,  235 
collecting  serum,  239 
immunizing  animals,  236 
rectal  method  of  administering,  759 
sequels,  764 

serum  sickness  from,  764 
standardizing,  240 

experimental  work,  911 
subcutaneous  injection,  759 
total  amount  to  be  administered,  764 
treatment  of  relapses  with,  764 
unit  of,  252 
value,  765 
bacillus,  113 

virulence  and  toxicity,   method  of 

testing,  894 

Behring's    method    of    immunization 
against,  769 


INDEX 


955 


Diphtheria,  diphtherin  in,  652 
nasal,  dosage  of  antitoxin  in,  762 
nature  of,  756 

of  eye,  dosage  of  antitoxin  in,  763 
of  vulva,  dosage  of  antitoxin  in,  763 
of  wounds,    dosage   of    antitoxin  in, 

763 
prophylactic    immunization    against, 

767 

serum  treatment,  754.     See  also  Diph- 
theria antitoxin. 

tonsillar,  dosage  of  antitoxin  in,  762 
toxin,  113 

experimental  work,  893 
limes  death  dose,  240 

zero  dose,  241 

method  of  testing  virulence,  114 
production  of,  235 
reaction  in  horses,  238 
standardizing,  115 
testing,  236 
toxoid,  117 

treatment  of,  756.       See  also  Diph- 
theria antitoxin. 
Diphtherin,  652 
Diplococcus  pneumonias,  808 
Disease,  antiferments  in,  257 

bacteriolytic  serums  in  treatment  of, 

381 
diagnosis  of,  Pfeiffer  bacteriolytic  test, 

370 

ferments  in,  258 
Pfeiffer's  bacteriolytic  test  in  diagnosis 

of,  370 

production  of,  108 
sero-enzymes  in,  277 
Dixon's  tuberculin,  dose  of,  727 

preparation,  719 
Dog,  anaphylaxis  in,  576 
intravenous  inoculation,  62 
obtaining  large  amounts  of  blood  from, 

51 

small  amounts  of  blood  from,  51 
serum,   normal,   non-specific   comple- 
ment fixation  by,  419 
Donath    and   Landsteiner's   method    of 
serum  diagnosis  of  paroxysmal  hemo- 
globinuria,  401 
Dose,  intoxicating,  576 
Dourine,    complement-fixation   test   in, 

515 

Drug  fastness,  849 
idiosyncrasy,  616 

Duhring's  disease,  salvarsan  in,  881 
modification  of  Wassermann  reaction, 

485 

Dysentery,  agglutination  test  in,  292 
antitoxin,  782 

administration  and  uses,  782 
bacillary,  vaccine  therapy,  713 
serum  treatment,  782 

results,  783 
toxin,  118 

experimental  work,  896 
vaccination,  699 


theory,   com- 


EAR,  tuberculosis  of,  tuberculin  test  in 

diagnosis  of,  631 
treatment,  731 

Echinococcus  disease  of  liver,  comple- 
ment-fixation test  in,  524 
miostagmin  reaction  in,  566 
Ecthyma,  vaccine  therapy,  705 
Eczema,  vaccine  therapy,  705 
Effusions,    non-tuberculous,    autoserum 

treatment,  843 

Egg-albumen  idiosyncrasy,  616,  652 
Ehrlich's  method  of  serum  diagnosis  of 

paroxysmal  hemoglobinuria,  401 
theory  of  immunity,  148 
and  Metchnikoff's 

patibility,  157 
therapia  magna  sterilisans,  851 
Electric  centrifuge,  18 
Emulsion,  bacterial,  in  opsonic  index,  197 
Emulsions,  543 
Endemic  infection,  82 
Endocarditis,  antistreptococcus  serum  in, 

817 

ulcerative,  vaccine  therapy,  713 
Endocellular  complement,  352 
Endocomplement,  352 
Endogenous  infection,  85,  86 
Endolysins,  185,  361 
Endotoxic   substances,    method   of   ob- 
taining, 122 

Endotoxins,  109,  122,  184 
as  anaphylactogens,  584 
experimental  work,  899 
influence  of  bacteriolysins  on,  362 
method  of  obtaining,  122 
nature  of,  123 

Enteritis  anaphylactica,  576 
Enzootic  infection,  82 
Enzymes,  254 

sero-,  in  disease,  277.      See  also  Sero- 
enzymes. 

Eosin-selenium  compound  in  cancer,  888 
Epidemic  cerebrospinal  meningitis,  treat- 
ment, 790 
infection,  82 

Epilepsy,  Wassermann  reaction  in,  490 
Epiphanin  reaction,  559 
in  cancer,  563 
in  malignant  disease,  563 
in  pregnancy,  563 
in  sarcoma,  563 
in  syphilis,  562 
principle,  559 
reading  results,  562 
specificity,  560 
Epitheliotoxin,  538 
Epizootic  infection,  82 
Erysipelas,  antistreptococcus  serum  in, 

817 

autoserum  treatment,  835 
vaccine  therapy,  706 
Erythrocytes  for  Wassermann  reaction, 

440 

method  of  determining  resistance,  402 
obtaining  of,  28 


956 


INDEX 


Erythrocytes,  resistance  of,  to  salt  solu- 
tion, experiemntal  work,  923 
washing  of,  28 

Exhaustion  theory  of  immunity,  146 
Exogenous  infection,  85 
Experimental  immunity,  890 

infection,  890 
Experiments,  animal,  891 
Exposure  as  predisposing  to  infection, 

103 
Eye,  diphtheria  of,  dosage  of  antitoxin 

in,  763 
tuberculosis    of,    tuberculin    test    in 

diagnosis  of,  631 
treatment,  731 


FAMILIAL  susceptibility,  101 
Fastigium  period  of  infection,  137 
Fastness,  drug,  849 
Feces,  bacteria  recovered  from,  bacterio- 

lytic  test  for  identification  of,  365 
Ferment  reactions,  238 
Ferments,  254 

and  toxins,  similarity  between,  254 

bacterial,  254 

experimental  work,  914 

in  disease,  258 

in  pregnancy,  258 

protective,  260 

Fever  and  chills  after  intravenous  in- 
jection of  salvarsan,  867 

of  infection,  139 

protein,  139 

Filariasis,  salvarsan  in,  881 
Film,  blood,  for  phagocytic  counts,  202 

method  of  preparing,  201 
Filter,  78 

Berkefeld,  77 

cleaning  and  care  of,  78 

Uhlenhuth,  328 
Filtration,  bacteria-free,  preservation  of 

serum  by,  76 

Finger  pricking,  method  of,  31 
Fixateur.    See  Amboceptor. 
Fixator,  344 

Flexner  and   Amoss   method   for   rapid 
production  of  antidysenteric  serum, 

and  Jobling's  method  of  preparing  anti- 

meningococcus  serum,  791 
Floccule-forming  precipitin  tests,  321 
Focal  infection,  94 
Fomites,  84 
Food    and    serum    hypersensitiveness, 

allergic  reactions  in,  652 
anaphylaxis,  615 
cutaneous  test,  616 
diagnosis,  616 
intracutaneous  test,  616 
treatment,  616 
Foods,  bacteria  in,  85 

idiosyncrasy,  615,  652 
Fordyce's   technic   of    intraspinous   in- 
jection of  salvarsanized  serum,  877 


Forensic  blood  test,  531 
Fornet's  ring  test  for  syphilis,  321 
Frambesia,  luetin  reaction  in,  646 

salvarsan  in,  880 

Wassermann  reaction  in,  491 
Freezing  serum,  preservation  by,  78 
Freund  and  Kaminer's  cytolytic  cancer 
diagnosis,  541 

theory  of  anaphylaxis,  592 
Fribo;  79 
Friedmann's  treatment  of  tuberculosis, 

722 

Fruits,  idiosyncrasy,  615 
Fulminating  infection,  138 
Furunculosis,  antistaphylococcus  serum 
in,  819 

vaccine  therapy,  704 


GALEOTTI  and  Lustig's  plague  vaccine, 

695 

Gastrotoxin,  539 
Gay  and  Ayer's  method  of  titration  of 

bacteriolytic  complement,  356 
and  Clay  pool's  sensitized  typhoid  vac- 
cine, 691 
typhoidin,  650 
and  Southard's  theory  of  anaphylaxis, 

592 
Gel,  definition,  544 

test  of  Donagh  for  syphilis,  323 
Genital  organs  as  portal  of  entrance  for 

bacteria,  89 
Genito-urinary  diseases,  vaccine  therapy, 

706 

tuberculosis,  tuberculin  test  in  diag- 
nosis of,  631 
treatment,  732 

Glanders,  agglutination  reaction  in,  293 
complement-fixation  test  in,  511 
mallein  reaction  for,  647.       See  also 

Mallein  reaction. 
Glandular  tuberculosis,  tuberculin  test 

in  diagnosis  of,  631 
Globulins,   Noguchi's  butyric-acid  test 

for,  322 

Goats,  intravenous  inoculation,  62 
Gonococcal  infections,  serum  treatment, 

818 
Gonococcus    antigen,    titration    of,    ex- 

perimenta  work,  936 
complement-fixation  test,  503 
antigen  for,  505 
experimental  work,  936 
hemolytic  systems,  504 
practical  value,  509 
specificity,  509 
technic,  504 
using  one-tenth    usual    amounts, 

.508 
infections,  allergic  reaction  in,  652 

complement  fixation  in,  503 
Gonorrheal  arthritis,   autoserum  treat- 
ment, 836 
vaccine  therapy,  707 


INDEX 


957 


Graduated  pipets,  21 

Gravity  method  of  intravenous  injection 

of  salvarsan,  863 
of  serum,  743 

of  subdural  injection  of  serum,  749 
Group  immune  bodies,  345 

precipitins,  315,  317 
Gruber-Widal  reaction  in  typhoid  fever, 

281,  290,  295,  299 
experimental  work,  916 
Gruber's  theory  of  mechanism  of  ag- 
glutination, 284 
Guinea-pig,  anaphylaxis  in,  573 

complement   serum  for  Wassermann 

reaction,  436 

intracardial  inoculation,  62 
intraperitoneal  inoculation,  64 
intravenous  inoculation,  57 
obtaining  large  amount  of  blood  from, 

47 
small  amount  of  blood  from,  41 


HAFFKINE'S  plague  vaccine,  dosage,  696 
effects,  696 
preparation,  695,  697 
results,  696 

Hamburger  and  Moro's  theory  of  anaphy- 
laxis, 591 

Hamman    and    Wolman's    method    of 
preparing    tuberculin    for     sub- 
cutaneous tuberculin  test,  633 
plan  of  dosage  for  tuberculin  in  sub- 
cutaneous test,  634 

Hansen  and  Kitchens'  method  of  pre- 
paring antimeningococcus  antigen,  793 
Haptines,  149 

Haptophore  group  of  toxin,  112 
Hay-fever,  252,  615 
antitoxin,  786 
serum  treatment,  786 
toxin  of,  120 
vaccine  therapy,  708 
Headache  after  injection  of  salvarsan, 

866 

Heart  failure  cells,  180 
Hecht-Weinberg-Gradwohl  modification 

of  Wassermann  reaction,  482 
Hemagglutination,  microscopic,  method 

of  Brem,  312 
Hemagglutinins,  286 

experimental  work,  919 
Hemocytometer  chamber,  counting  bac- 
terial vaccines  with,  215 
Hemoglobinuria,  paroxysmal,  serum  di- 
agnosis, 401 
Hemolysins,  156,  340,  383 

action  of,  based  on  colloidal  reaction. 

551 
and  bacteriolysins,  analogy  between, 

389 

definition,  384 

experimental  production,  892 
history,  383 
immune,  385 


Hemolysins,  immune,  method  of  titra- 

tion,  397 

production  of,  393 
methods  for  removing,  from  serum, 

400 
natural,  391 

experimental  work,  928 

method  of  determining,   in  serum, 

400 

removal  of,  experimental  work,  928 
nature,  385 
nomenclature,  385 
normal,  390 

practical  applications,  395 
preservation  of,  in  dried  paper  form, 

80 
production  of,  71 

combined     intravenous-intraperito- 

neal  method,  73 
intraperitoneal  method,  72 
intravenous  method,  72 
properties,  393 
sources,  394 
specific,  385 
specificity,  390 
Hemolysis,  383 
and   bacteriolysis,    analogy   between, 

389 

non-specific,  402 
relation  of  lipoids  to,  554 
role  of  amboceptor  and  complement 

in,  experimental  work,  926 
serum,  experimental  work,  924 

quantitative   factors,    experimental 

work,  925 
venom,  405,  554.       See  also  Venom 

hemolysis. 
Hemolytic  amboceptor,  341,  384 

and  complement,   quantitative  re- 
lationship, 396 

for  Noguchi's  modification  of  Was- 
sermann reaction,  476 
for  Wassermann  reaction,  439 
preservation  of,  80 
titration  of,  347 

experimental  work,  925 
complement,  experimental  work,  929 
titration  of,  356 

experimental  work,  931 
influence  of  acids  and  alkalis,  experi- 
mental work,  924 
jaundice,  395 
titration  of  antigens  in  Wassermann 

reaction,  454 
Hemophilia,   normal   serum   treatment, 

829 
Hemopsonins,  191 

experimental  work,  908 
Hemorrhage,  normal  serum  treatment. 

829 

Hemorrhagin,  786 
Hemotoxin,  119 

cobra,  122 

Hens,  anaphylaxis  in,  577 
Hepatotoxin,  539 


958 


INDEX 


Herman-Perutz  test  for  syphilis,  322 
Herxheimer-Jarisch  reaction,  874 
Heterolysins,  385 

Kitchens  and  Hansen's  method  of  pre- 
paring antimeningococcus  antigen, 
793 

syringe,  241 

Hodgkin's  disease,  salvarsan  in,  881 
Hog  cholera  serum,  production,  784 
standardization,  784 
treatment,  784 
obtaining  large  amount  of  blood  from, 

50 
Hopkins'  method  of  counting  bacterial 

vaccines,  216 

tube  for  standardizing  bacterial  vac- 
cine, 217 

Horse,  anaphylaxis  in,  577 
asthma,  615,  652 
diphtheria  toxin  reaction  in,  238 
intravenous  inoculation,  61 
obtaining  large  amount  of  blood  from, 

51 

small  amount  of  blood  from,  51 
syphilis,  complement-fixation  test  in, 

515 
Humoral  theory  of  anaphylaxis,  584,  591 

of  immunity,  145,  148 
Hydatid    disease,    complement-fixation 

test  in,  524 

Hydrocele,  autoserum  treatment,  842 
Hydrocephalus  in  meningococcus  menin- 
gitis,  effect  of  serum  treatment  on, 
799 

Hydrophobia,  681.     See  also  'Rabies. 
Hypersensitiveness,  567,  614,  652.     See 

also  Idiosyncrasy. 
Hypothesis  of  Bail,  124 
of  Welch,  104 


IDIOSYNCRASY,  567,  614 
buckwheat,  615 
drugs,  616 

egg-albumin,  616,  652 
foods,  615,  652 
fruits,  615 
hay-fever,  615 
horse  asthma,  615,  652 
iodoform,  616 
pork,  615 
strawberries,  615 
vegetables,  615,  652 
Immune  agglutinins,  281 
bodies,  341,  384 

group,  345 

partial,  345 
hemolysins,  385 

method  of  titration,  397 

production  of,  393 
opsonins,  experimental  work,  908 

production  of,  69 
precipitins,  316 
serums  and  normal  serums,  difference 

between,  346 


Immune  serums,  methods  for  making,  66 
precipitin,  70 
preservation  of,  76 

in  dried  paper  form,  80 
in  fluid  form,  by  bacteria-free  fil- 
tration, 76 
by  freezing,  78 
with  antiseptics,  76 
in  living  animal,  80 
in  powder  form,  79 
standardization  of,  complement-fix- 
ation test  in,  524 
Immunity,  140 
acquired,  172 
active,  172 

by  vaccination,  173 
causes  of,  172 
experimental  work,  903 
antibacterial,  173 
antitoxic,  173 
experimental  work,  903 
passive,  174 
antitoxic,  175 
experimental  work,  904 
Vaughan's  theory,  176 
active,  65 

agglutinins  in,  role  of,  289 
anaphylaxis  in  relation  to,  602 
antibacterial,  736 

experimental  work,  902 
antitoxic,  736 
antitoxin,  natural,  171 
athreptic,  172 
cellular  theory,  145 
cytotoxins  in,  role  of,  540 
definition  of,  142 
Ehrlich's  theory,  148 

and  Metchnikofif's  theory,   com- 
patibility, 157 
exhaustion  theory,  146 
experimental,  890 
history  of,  142 
humoral  theory,  145,  148 
individual,  168 
Metchnikoff's  theory,  146 

and    Ehrlich's    theory,    compati- 
bility, 157 
natural,  167 
antitoxin,  171 
causes  of,  169 

influence    of    temperature    on,    ex- 
perimental work,  903 
phagocytosis  in,  170 

experimental  work,  902 
relative    factors    in,-    experimental 

work,  903 

Vaughn's  theory,  176 
opsonins  in,  193 
passive,  734 

varieties  of,  735 
phagocytosis  theory,  146 

and  side-chain  theory,   compati- 
bility, 157 

revised  theory  and  role,  188 
precipitins  in,  role  of,  319 


INDEX 


959 


Immunity,  racial,  168 
reaction,  606 

colloidal  reaction  and,  analogy  be- 
tween, 548 

relation  of  colloids  to,  543 
of  infection  to,  83 
of  lipoids  to,  553 
of  phytotoxins  to,  119 
retention  theory,  146 
side-chain  theory,  148 

and  phagocytic  theory,  compati- 
bility, 157 
species,  168 
theories  of,  140,  146 
types  of,  167 
Vaughan's  theory,  175 
Immunization,  active,  654 
contraindications  to,  667 
for  prophylaxis,  659 
for  therapeutic  purposes,  702 
for  treatment  of  disease,  660 
history  of,  654 
mechanism  of,  659 
negative  phase,  666 
of  animals,  700 
general  technic,  66 
methods  for  effecting,  65 
antibacterial,  736,  787 
antitoxic,  736,  754 
Behring's  method,  against  diphtheria, 

769 

curative,  734,  735 
passive,  733 

contraindications  to,  738 
indications  for,  736 
purposes  of,  734 
prophylactic,  618,  668,  734.     See  also 

Vaccination. 

in  paratyphoid  fever,  693 
in  pertussis,  700 
in  pneumonia,  694 
in  typhus  fever,  694 
therapeutic,  618 
Impetigo,  vaccine  therapy,  705 
Incubation  period  of  infection,  136 
Index,  opsonic,  194.      See  also  Opsonic 

index. 

phagocytic,  203 
Individual  immunity,  168 

predisposition,  101 

Infantile     paralysis,     treatment      with 
specific  serum  of  convalescents  and 
with  normal  serum,  826 
Infected  wounds,  vaccine  therapy,  703 
Infection,  81 
acute,  138 

sero-enzymes  in,  278 
vaccine  therapy,  713 
aggressins  in  relation  to,  104 
anaphylaxis  in  relation  to,  602 
antenatal,  90 

avenue  of,  tissue  susceptibility  and,  98 
avenues  of,  86 
chronic,  138 
complications,  138 


Infection,  course,  136 

cryptogenic,  92 

defensive  mechanism  of  micro-organ- 
ism in  relation  to,  103 

definition  of,  81 

diet  as  predisposing  to,  102 

endemic,  82 

endogenous,  85,  86 

enzootic,  82 

epidemic,  82 

epizootic,  82 

exogenous,  85 

experimental,  890,  892 

exposure  as  predisposing  to,  103 

fever  of,  139 

focal,  94 

vaccine  therapy,  704 

fulminating,  138 

general  susceptibility  in  relation  to, 
100 

gonococcus,  allergic  reaction  in,  652 
serum  treatment,  818 

grades  of,  138 

intoxications  as  predisposing  to,  102 

malignant,  138 

malnutrition  as  predisposing  to,  102 

mechanism  of,  94 

mixed,  106 

absorption  agglutination  test  in,  309 

morbid  conditions  as  predisposing  to, 
103 

morphologic  changes  of  micro-organ- 
isms in  relation  to,  103 

numeric  relationship  of  bacteria  to,  99 

pandemic,  82 

period  of  convalescence,  138 
of  decline,  137 
of  fastigium,  137 
of  high  fever,  137 
of  incubation,  136 
of  prodromal  symptoms,  137 

physiologic  changes  of   micro-organ- 
isms in  relation  to,  103 

pneumococcus,  chemotherapy  in,  885 
serum  treatment,  807 

precipitation  of  extracellular  toxins, 
111 

previous,  as  predisposing  to  infection, 
102 

relapse  of,  138 

relation  of,  to  immunity,  83 

remittent,  138 

selective  tissue  affinity,  99 

sequels  of,  138 

sources  of,  83 

sporadic.  82 

stages  of,  136 

staphylococcus,  serum  treatment,  819 

streptococcus,  serum  treatment,  813 

systemic  reaction  to,  139 

trauma  as  predisposing  to,  103 

with  animal  parasites,  134 
modes,  134 

wound,    antistreptococcus   serum   in, 
817 


960 


INDEX 


Infectious  diseases,  84 

accelerated  anaphylaxis  reaction  in, 

605 

acute,  autoserum  treatment,  835 
anaphylaxis,  torpid,  early  reaction 

in,  605 
immediate  anaphylaxis  reaction  in, 

605 

production  of,  108 
relation  of  anaphylaxis  to,  603 
treatment   with   specific   serum   of 
convalescents   and   with   normal 
serum,  825 

Infestation,  definition  of,  81 
Infestment,  definition  of,  81 
Inflammation,  behavior  of  leukocytes 

in,  182 

Influenza,  autoserum  treatment,  836 
Influenza!  meningitis,  serum  treatment, 

802 
Inoculation,    animal,    53.         See    also 

Animal  inoculation. 

Insects,  suctorial,  transmission  of  bac- 
teria by,  86 
Intel-body,  156,  341 
Intestine,    tuberculosis    of,    tuberculin 

treatment,  731 
Intoxicating  dose,  572 
Intoxications  as  predisposing  to  infec- 
tion, 102 
Intrabronchial  route  for  tuberculin  treat-  4 

ment,  729 
Intracardial  inoculation  of  animals,  62 

of  guinea-pigs,  62 
Intracutaneous  tuberculin  test  of  Man- 

tous,  628,  636 
of  Mendel,  628,  636 

in  animals,  642 
Intraf ocal  route  for  tuberculin  treatment, 

729 

Intramuscular  injection  of  animals,  56 
of  diphtheria  antitoxin,  759 
of  neosalvarsan  in  syphilis,  875 
of  salvarsan  in  syphilis,  875 
of  serum,  treatment,  742 
of  tetanus  antitoxin,  774 
Intraneural  injection   of   tetanus   anti- 
toxin, 775 

Intraperitoneal  and  intravenous  methods 
combined  for  production  of  hemol- 
ysins,  73 

inoculation  of  animals,  64 
of  guinea-pig,  64 
of  rabbit,  64 
method  of  production  of  agglutinins, 

69 

of  hemolysins,  72 

Intravenous  and  intraperitoneal  methods 
combined  for  production  of  hem- 
olysins, 73 

administration  of  neosalvarsan  in  con- 
centrated solution,  866 
of  salvarsan  in  concentrated  solu- 
tion, 865 
injection  of  animals,  56 


Intravenous  injection  of  antimeningococ- 

cus  serum,  796 

of  antipneumococcus  serum,  812 
of  antistreptococcus  serum,  816 
of  diphtheria  antitoxin,  759 
of  dog,  62 
of  goats,  62 
of  guinea-pig,  57 
of  horse,  61 
of  mice,  60 

of  neosalvarsan  in  syphilis,  858 
after-care,  866 
apparatus,  863 
dosage,  859 
frequency,  859 
gravity  method,  863 
intensive  treatment,  859 
preparation  of  patient,  859 

of  solution,  862 
of  rabbit,  56 
of  rat,  60 

of  salvarsan  in  syphilis,  858 
after-care,  866 
after-effects,  866 
apparatus,  863 
chills  and  fever  after,  867 
diarrhea  after,  867 
dosage,  859 
frequency,  859 
gravity  method,  863 
headache  after,  866 
Herxheimer  reaction  after,  875 
intensive  treatment,  859 
Jarisch-Herxheimer       reaction 

after,  874 
preparation  of  patient,  859 

of  solution,  860 
of  serum,  gravity  method,  743 
syringe  method,  742 
treatment,  742 
of  sheep,  62 

of  tetanus  antitoxin,  774 
method  of  producing  hemolysins,  72 

serum  precipitins,  70 
route  for  tuberculL  treatment,  729 
Invasion,  82 

lodoform  idiosyncrasy,  616 
Isocytotoxins,  538 
Isohemagglutinins,  286 
relation  of,  to  blood  transfusion,  286 
tests  for,  before  transfusion  of  blood, 

311 
Isohemolysins,  392 

tests  for,  before  transfusion  of  blood, 

311 

Isolysins,  385 
Isoprecipitin,  317 

Izar  and  Ascoli's  miostagmin  test,  563. 
See  also  Miostagmin  reaction. 


JAFFE  and  Topfer's  method  of  measuring 

bactericidal  power  of  blood,  374 
Jarisch-Herxheimer  reaction,  874 
Jaundice,  hemolytic,  395 


INDEX 


961 


Jenner  on  vaccination,  143,  669 
Jennerian  vaccination,  173 
Jobling  and  Flexner's  method  of  prepar- 
ing antimeningococcus  serum,  791 
Joints,  tuberculosis  of,  tuberculin  test 

in  diagnosis  of,  631 
treatment,  731 


KAMINER  and  Freund's  cytolytic  cancer 

diagnosis,  541 

Keidel  tube  for  collecting  blood,  35,  36 
Keratosis  follicularis,  salvarsan  in,  881 
Koch's    subcutaneous    tuberculin    test, 

628,  633 
in  animals,  641 

Kolle  and  Pfeiffer's  macroscopic  agglu- 
tination test,  305 
and  Strong's  plague  vaccine,  695 
Kolle's  cholera  vaccine,  preparation,  697 
method  of  counting  bacterial  vaccines, 

216 

antipneumococcus  serum,  811 
antistreptococcus  serum,  816 
plague  serum,  823 
plague  vaccine,  preparation,  695 
Kolmer's  method  of  testing  virulence  and 

toxicity  of  diphtheria  bacilli,  114 
Korte  and  Stern's  method  of  measuring 
bactericidal  power  of 
blood,  371 

experimental  work,  939 
Kraus'  anticholera  serum,  823 
Kretz,  paradox  of,  584 


LACTOSERUM,  315 

experimental  work,  922 
production  of,  71 

Landsteiner  and  Donath's  method  of 
serum  diagnosis  of  paroxysmal  hemo- 
globinuria,  401 

Lange's  colloidal  gold  reaction,  555 
practical  value,  558 
preparation   of   colloidal   gold, 

556 

principles,  555 
types  of  reaction,  557 
Larynx,  tuberculosis  of,  tuberculin  test 

in  diagnosis  of,  631 
Law,  Colics',  Wassermann  reaction  and, 

489 
Profeta's,  Wassermann  reaction  and, 

490 

Lecithid,  554 
cobra,  352 
Lecithin,  406.  554 

and  cholesterin  for  Wassermann  re- 
action, 447 
mono-fatty-acid-,  406 
Leistenkern,  149 
Leitenketter,  149 
Leprosy,  allergic  reactions  in,  653 
autoserum  treatment,  776 
luetin  reaction  in,  646 

61 


Leprosy,  salvarsan  in,  881 

Wassermann  reaction  in,  491 
Leukins,  360 
Leukocidin,  119 
Leukocytes,  behavior  of,  in  inflammation, 

in  quantitative  estimation  of  bacterio- 

tropins,  204 
obtaining  of,  28 
washed,  in  opsonic  index,  198 
Leukocytic  extracts,  360 

preparation,  361 
Leukotoxins,  538 
Lichen  planus,  salvarsan  in,  881 
Limes  death  dose  of  diphtheria  toxin,  240 

zero  dose  of  diphtheria  toxin,  241 
Lipoid  anaphylaxis,  580 
Lipoids,  162,  428 

acetone-insoluble,  for  Wassermann  re- 
action, 446 

relation  of,  to  hemolysis,  554 
to  immunity,  553 
to  Wassermann  reaction,  555 
Liver,  echinococcus  disease  of,  comple- 
ment fixation  test,  524 
syphilitic,  alcoholic  extracts,  for  Was- 

sermann  reaction,  444 
aqueous  extracts,  for  Wassermann 

reaction,  443 
Lobar  pneumonia,  nature,  808 

serum  treatment,  807 
Locomotor  ataxia,  Lange's  colloidal  gold 

reaction  in,  559 
Wassermann  reaction  in,  488 
Looped  pipets,  20 
making  of,  20 
Luetin,  preparation  of,  643 
reaction,  642 

in  frambesia,  646 
in  leprosy,  646 
in  yaws,  646 

method  of  application,  643 
negative,  644 
normal,  644 
papular  form,  644 
positive,  644 
practical  value,  646 
preparation  of  luetin,  643 
pustular  form,  645 
results,  646 
torpid  form,  645 
Lumbar  puncture,  37 

after-treatment  of  patient,  41 
anesthesia  in,  39 
contraindications,  37 
disposal  of  fluid,  41 
injection  of  antimeningococcus  se- 
rum by,  795 
of  neosalvarsan  by,  875 
of  serum  by,  746 
of  tetanus  antitoxin  by,  775 
preparation  of  patient,  38 
technic,  39 
value,  37 
Lupus  vulgaris,  salvarsan  in,  881 


962 


INDEX 


Lustig   and    Galeotti's  plague  vaccine, 

695 
Lustig's    method    of    preparing    plague 

serum,  823 
Lymphocytosis,  180 
Lysins,  73,  156 


MACKENZIE  and  Browning's  modification 

of  Wassermann  reaction,  484 
test  for  estimating  amount  of  com- 
plement absorbed  in  Wassermann 
reaction,  458 
Macrocytase,  159,  185 
Macrophages,  147,  179 

experimental  work,  905 
Malaria,  salvarsan  in,  881 

Wassermann  reaction  in,  491 
Malignant  disease,  chemotherapy  in,  887 

epiphanin  reaction  in,  563 
infection,  138 
Mallein  reaction,  647 
ophthalmic,  648 
preparation  of  mallein,  647 
subcutaneous,  648 

Malnutrition  as  predisposing  to  infec- 
tion, 102 
Malta  fever,  agglutination  test  in,  292 

autoserum  treatment,  836 
Mantoux's     intracutaneous     tuberculin 

test,  628,  636 

Maragliano's  tuberculosis  serum,  824 
Marasmus,  autoserum  treatment,  843 
Marcus  modification  of  M  tiller  and  Joch- 
mann's  method  of  testing  blood-serum, 
experimental  work,  914 
Markl's    method    of    preparing    plague 

serum,  823 

Marmorek's  tuberculosis  serum,  824 
McDonagh  gel  test  for  syphilis,  323 
Measles,  effect  of,  on  tuberculin  reaction, 

627 
Meat   adulteration,    detection,    biologic 

blood  test  for,  333 
technic,  335 

Meats,    identification   of,    complement- 
fixation  test  for,  531 
Mendel's  intracutaneous  tuberculin  test, 

628,  636 
in  animals,  642 
Meningitis,  cerebrospinal,  agglutination 

test  in,  292 

precipitin  test  in  diagnosis,  320 
serum  treatment,  788 
vaccination  against,  699 
influenza!,  serum  treatment,  802 
meningococcus,    epidemic,    treatment 

of,  790 

nature  of,  790 
prophylactic  immunization  in,  801, 

serum  treatment,  788 

cases  of  posterior  basal  menin- 
gitis, 798 
with  dry  canal,  798 


Meningitis,  meningococcus,  serum  treat- 
ment, cases  with  thick  elastic 
exudate,  798 
chronic  cases,  799 
results,  799 
serum  sickness  in,  799 
subacute  cases,  799 
pneumococcus,  804 

serum  treatment,  804,  805 
tuberculous,  autoserum  treatment,  843 
tuberculin    test    in    diagnosis    of, 

632 

treatment,  731 
Meningococcus      meningitis,    epidemic, 

treatment  of,  790 
nature,  790 
prophylactic  immunization  in,  801, 

802 
serum  treatment,  788 

cases  of  posterior  basal  menin- 
gitis, 798 

with  dry  canal,  798 
with   thick   elastic   exudate, 

798 

chronic  cases,  799 
results,  799 
serum  sickness  in,  799 
subacute  cases,  799 

Mental  deficiency,  congenital,  Wasser- 
mann reaction  in,  490 
diseases,  sero-enzymes  in,  278 
Mercury   treatment    of    syphilis,    effect 

of,  on  Wassermann  reaction,  492 
Mesenteric  glands,  tuberculosis  of,  tu- 
berculin treatment,  731 
Metchnikoff's  theory  of  immunity,  146 
and  Ehrlich's  theory,  compatibil- 
ity, 157 

of  phagocytosis,  178 
Meyer  and  Bergmann's  antitrypsin  test, 

264 
Mice,  intravenous  inoculation,  60 

white,  anaphylaxis  in,  576 
Microcytase,  159,  185 
Micro-organisms,  83.     See  also  Bacteria. 

selective  affinity,  99 
Microparasites,  complement-fixation  test 

in  differentiation,  502 
Microphages,  146,  179 

experimental  work,  905 
Milk,  bacteria  in,  85 

precipitins,  experimental  work,  922 

production  of,  71 
Miostagmin  reaction,  563 
experimental  work,  940 
in  cancer,  565,  566 
in  echinococcus  disease,  566 
in  paratyphoid  fever,  566 
in  syphilis,  566 
in  tuberculosis,  566 
in  typhoid  fever,  566 
practical  value,  566 
principles,  563 
technic,  564 
Mixed  infection,  106 


INDEX 


963' 


Monkey,  obtaining  large  amount  of  blood 

from,  50 
small  amount  of  blood  from,  50 

Mono-fatty-acid-lecithin,  406 

Morbid  conditions  as  predisposing  to 
infection,  103 

Moro  and  Hamburger's  theory  of  ana- 
phylaxis,  591 

Moro's  percutaneous  tuberculin  test, 
628,  640 

Much's  psycho-reaction,  410 
technic,  410 

Mule  serum,  normal,  non-specific  com- 
plement-fixation by,  419 

Miiller  and  Jochmann's  method  of  test- 
ing blood-serum,  Marcus  modification, 
experimental  work,  914 

Multiform  rashes  in  serum  disease,  611 

Multiple  pipet,  23 

Mycosis  fungoides,  salvarsan  in,  881 


NASAL  diphtheria,  dosage  of  antitoxin 

in,  762 
Nastin,  162 

Negri  bodies  in  rabies,  682 
Neisser    and    Wechsberg's    method    of 
measuring  bactericidal  power  of  blood, 
371 

Neosalvarsan  in  syphilis,  852 
administration,  858 
effect  of,  on  Wassermann  reaction, 

495 

history,  852 
in  concentrated  solution,  intravenous 

administration,  866 
intramuscular  injection,  875 
intravenous  injection,  858.   See  also 
Intravenous  injection  of  neosalvar- 
san. 
preparation  of  dilute  solution  for 

intravenous  injection,  862 
subdural  injection,  875 
Wile's  technic,  876 
value,  879 
properties  of,  856 
Nephritis,  contraindications  to  vaccines 

in,  668 

normal  serum  treatment,  834 
Nephrotoxic  serum,  production  of,  73 
Nephrotoxin,  538 

action  of,  experimental  work,  940 
Neufeld's    quantitative    estimation    of 

bacteriotropins,  203 
controls,  205 
culture,  205 
leukocytes,  204 
precautions,  206 
readings,  205 
serum,  204 

Neurorrhyctes  hydrophobias,  682 
Neurotoxin,  540 

New  York  Board  of  Health  outfit  for 
collecting  blood  for  Wassermann  re- 
action, 434 


Ninhydrin,  267 

Noguchi's  butyric-acid  test  for  protein, 

322 
globulin  reaction,  experimental  work, 

922 
luetin  reaction,  642.     See  also  Luetin 

reaction. 

method  of  preparing  cowpox  virus,  674 
modification  of  Wassermann  reaction, 

475 
antigen  for,  478 

titration  of,  479 
complement  for,  476 
experimental  work,  935 
fluid  to  be  tested,  480 
hemolytic  amboceptor  for,  476 
human  corpuscles  for,  476 
technic,  476 
test,  480 

Nolf's  theory  of  anaphylaxis,  593 
Nucleoproteins,  production  of,  74 
Nuttall's    method    of    obtaining    large 
amount  of  blood  from  rabbit,  43 


OPHTHALMIC  mallein,  preparation,  648 

reaction,  648 

reaction  in  typhoid  fever,  649 
Opsonic  index,  194 

as  diagnostic  procedure,  207 

as  guide  to  size  and  frequency  of 
doses  of  bacterial  vaccines,  208 

bacterial  emulsion  in,  197 

collection  of  patient's  and  control 
serum,  196 

definition,  194 

determining,  experimental  work,  910 

in  prognosis,  208 

limitation,  195 

practical  value,  206 

precautions  in  technic,  196 

principle,  194 

purpose,  195 

technic,  196 

precautions  in,  196 

washed  leukocytes  in,  198 
Opsonification,  susceptibility  to,  192 
Opsonins,  147,  187,  190 

action   of,    mechanism,    experimental 

work,  908 
definition  of,  191 
effect  of,  on  bacteria,  192 
experimental  work,  891,  907 
history  of,  190 
immune,  experimental  work,  908 

production  of,  69 
in  immunity,  193 
nature  of,  191 

normal,  experimental  work,  907 
properties  of,  191 
source  of,  192 

specificity  of,  experimental  work,  909 
Oral  method  of  administering  diphtheria 

antitoxin,  759 
route  for  tuberculin  treatment,  728 


964 


INDEX 


Organotropism,  845 

Osmotic  pressure  of  colloids,  545 

O.  T.  tuberculin,  dose  of,  726 

preparation  of,  716 
Otitis  media,  vaccine  therapy,  711 
Overproduction  theory  of  Weigert,  150 
Overwork  as  predisposing  to  disease,  101 
Oxford  University  macroscopic  agglutin- 
ation test,  305 
technic,  305 


PALTAUF'S  theory  of  mechanism  of  ag- 
glutination, 284 
Pandemic  infection,  82 
Paradox  of  Kretz,  584 
Paralysis,  general,  Wassermann  reaction 

in,  487 

infantile,  treatment  with  specific  serum 
of  convalescents  and  with  normal 
serum,  826 

Paralytic  dementia,  Wassermann  reac- 
tion in,  487 
Parasites,  83 

animal,  aggressiveness  of,  135 
infection  with,  134 

modes,  134 

production  of  disease  by,  135 
half,  126 
partial,  126 
true,  125 

Parasitotrqpism,  845 
Parasyphilitic  diseases,  Wassermann  re- 
action in,  487 
Paratyphoid  fever,  miostagmin  reaction 

in,  566 
Oxford  University  agglutination  test 

in  diagnosis  of,  305 
prophylactic  immunization  in,  693 
Paresis,  Lange's  colloidal  gold  reaction 

in,  559 

Paroxysmal  hemoglobinuria,  serum  diag- 
nosis, 401 

Pasteur  on  immunity,  143,  144 
treatment  of  rabies,  intensive,  686 
mild,  687 
principle,  685 
results,  687 

Pasteurian  vaccination,  173 
Pearce's  method  of  producing  nephro- 

toxic  serum,  74 

Pellagra,  autoserum  treatment  in,  836 
salvarsan  in,  881 
Wassermann  reaction  in,  491 
Pelvic  tuberculosis,   tuberculin  test   in 

diagnosis  of,  631 
Percutaneous  tuberculin  test  of  Moro, 

628,  640 
Peritonitis,  tuberculous,  tuberculin  test 

in  diagnosis  of,  632 

Pertussis,  agglutination  reaction  in,  293 
complement-fixation  reaction  in,  521 
prophylactic  immunization  in,  700 
vaccine  therapy,  710 
Pfaundler's  reaction,  280 


Pfeiffer  and  Kolle's  macroscopic  agglu- 
tination test,  305 
Pfeiffer's  bacteriolytic  test,  364 
experimental  work,  938 
in  diagnosis  of  disease,  370 
technic,  369 
Phagocytes,  146,  178 

varieties,  179 
Phagocytic  index,  203 
Phagocytosis,  147,  177 

experimental  work,  905,  906 

history,  177 

in  immunity,  revised  theory  and  rdle 

of,  188 
in  natural  immunity,  170 

experimental  work,  902 
mechanism  of,  189 
MetchnikofFs  theory,  178 
relation  of  body-fluids  to,  186 

of  cell  types  to  infection,  180 
results  of,  184 
revised  theory  of,  188 
spontaneous,  190 
theory  of  immunity,  146 

and  side-chain  theory,  compatibil- 
ity, 157 
Phenomenon,    Arthus,    of    anaphylaxis, 

569 
Bordet-Gengou,  354,  413.       See  also 

Bordet-Gengou  phenomenon. 
Smith's,  of  anaphylaxis,  571 
Phlebotomy,  33 
in  children,  33 
Phogpsin,  182 

Physical  theory  of  anaphylaxis,  593 
Phytoprecipitin,  314 
Phytotqxins,  110,  119 
experimental  work,  898 
general  properties,  120 
relation  to  immunity,  120 
Pigeons,  anaphylaxis  in,  577 
Pipets,  18 

capillary,  for  counting  bacterial  vac- 
cine, 214 

for  opsonic  index  determination,  199 
making  of,  18 
method  of  sealing,  201 
.rubber  teats  for,  20 
Comer's  automatic,  219 
for  Wassermann  reaction,  431 
graduated,  21 
looped,  20 
making  of,  20 
multiple,  23 
sterilization  of,  22 
Piroplasma  bigeminum,  173 
Pirquet's  cutaneous  tuberculin  test,  628 

637 

in  animals,  642 
studies  on  anaphylaxis,  569 
Pityriasis  rubra,  salvarsan  in,  881 
Placenta  as  portal  of  entrance  for  bac- 
teria, 90 

bacteria  in,  86,  90 
Placental  blood,  method  of  obtaining,  37 


INDEX 


965 


Placental  serum,  method  of  obtaining, 

833 

Plague,  agglutination  test  in,  292 
serum  treatment,  822 
vaccination,  694 

dosage  of  vaccine,  696 
duration,  696 
effects,  696 

Haffkine's  vaccine,  695 
dosage,  696 
effects,  696 
results,  696 

Kolle  and  Strong's  vaccine,  695 
Kolle's  vaccine,  695 
Lustig  and  Galeotti's  vaccine,  695 
preparation  of  vaccine,  695 
results,  696 

Terni  and  Bandi's  vaccine,  696 
Plants,  higher,  toxins  of,  119 
Plasma,  blood,  obtaining  of,  30 
Pleurisy,  tuberculous,  autoserum  treat- 
ment, 841 

tuberculin  test  in  diagnosis  of,  632 
Pneumococci,  agglutination  test  in  dif- 
ferentiation, 308 

Pneumococcus,  Cole's  method  of  deter- 
mining type,  308 
infections,  chemotherapy  in,  885 

serum  treatment,  807 
meningitis,  805 

serum  treatment,  804,  805 
Pneumonia,  agglutination  test  in,  292 
autoserum  treatment,  836 
bacterial  vaccines  in,  712 
experimental,  892 
nature  of,  808 
precipitin  reaction  in,  321 
prophylactic  immunization  in,  694 
serum  treatment,  807,  828 

results,  812 

treatment  with  specific  serum  of  con- 
valescents and  with  normal  serum, 
826 

vaccine  therapy,  712 
Poison,  protein,  584 
Polariscope,  276 

Poliomyelitis,  acute  anterior,  treatment 
with  specific  serum  of  convalescents 
and  with  normal  serum,  826 
Pollen  antitoxin,  252,  786 

toxin,  120 
Polyceptor,  342 

Porges-Meier  test  for  syphilis,  321 
Pork  idiosyncrasy,  615 
Precipitate,  316 
Precipitation  of  colloids,  545,  546.    See 

Precipitin  test. 
Precipitin,  154,  314 

action  of,  based  on  colloidal  reactions, 

550 

bacterial,  experimental  work,  922 
precipitin  test  with,  320 
technic,  336 

E  reduction  of,  71 
nition,  314 


Precipitin,  experimental  production,  892 

work,  920 
formation,  317 
group,  315,  317 
history,  314 
immune,  316 
in  immunity,  role  of,  319 
milk,  experimental  work,  922 

production  of,  71 
nomenclature,  316 
normal,  316 
production  of,  70 

intravenous  method,  70 
properties,  316 

protein,  precipitin  test  with,  324 
reaction  in  pneumonia,  321 
serum,  production  of,  70 

titration,  experimental  work,  920 
specificity,  318 

experimental  work,  920 
structure,  316 
test,  floccule-forming,  321 
for  blood-stain,  326 

technic,  331 
for  detection  of  meat  adulteration, 

333 

technic,  335 
for   differentiation   of   human   and 

animal  blood,  326 
Fornet's,  for  syphilis,  321 
Herman-Perutz,  for  syphilis,  322 
mechanism,  316 
Porges-Meier,  for  syphilis,  321 
practical  applications,  320 
technic,  326 

with  bacterial  precipitins,  in  diag- 
nosis of  cerebrospinal  menin- 
gitis, 320 

practical  application,  320 
technic,  336 
with   protein  precipitins,   practical 

application,  324 
Precipitinogen,  316 

bacterial,  preparation  of,  336 
Precipitoid,  316,  317 
Predisposition,  101.     See  also  Suscepti- 
bility. 

Pregnancy,  Abderhalden's  serodiagnosis 
of,  265.        See  also  Abderhalden's 
serodiagnosis  of  pregnancy. 
allergic  reaction  in,  653 
epiphanin  reaction  in,  563 
ferments  in,  258 
toxicoses  of,  normal,  serum  treatment, 

833 
vomiting  of,  normal,  serum  treatment, 

833 

Preparator,  341 
Pricking  finger  method,  31 
Pro-agglutination,  304 

experimental  work,  919 
Pro-agglutinoids,  281 
Prodromal  symptoms  of  infection,  137 
Profeta's  law,  Wassermann  reaction  and, 
490 


966 


INDEX 


Prophylactic    immunization,    618,    667, 

668.     See  also  Vaccination. 
Protective  ferments,  260 

substances,  65,  734 

Proteids,  relation  of  antitoxins  to,  227 
Protein  anaphylactogens,  chemistry,  580 
bacterial,  109,  127 
action  of,  129 
experimental  work,  900 
nature  of,  128 
split,  128 

Vaughan's  theory,  130 
complement  fixation  in  differentiation 

of,  experimental  work,  937 
differentiation    by    complement    fixa- 
tion, 527 
fever,  139 

increased  amount,  Noguchi's  butyric- 
acid  test  for  detection,  322 
matrix  in  anaphylaxis,  587 
poison,  584 

precipitins,  precipitin  test  with,  324 
Protoxin,  116 
Provocatory  stimulation  of  Wassermann 

reaction,  495 

Psoriasis,  salvarsan  in,  881 
Psycho-reaction  of  Much,  410 

technic,  410 
Ptomai-ns,  109,  131 

experimental  work,  901 
Puerperal  sepsis,   antistreptococcus  se- 
rum in,  817 
vaccine  therapy,  713 
Pulmonary  tuberculosis,  tuberculin  test 

in  diagnosis  of,  630 
Pump,  suction,  29 
Puncture,  lumbar,  37.     See  also  Lumbar 

puncture. 

spinal,  37.    See  also  Lumbar  puncture. 
Pyelitis,  vaccine  therapy,  706 
Pyemia,  93 
Pyocyanase,  254 


RABBIT,  anaphylaxis  in,  575 
intraperitoneal  inoculation,  64 

method  of  production  of  agglutinins 

in,  69 

intravenous  inoculation,  56 
obtaining  large  amounts  of  i»lood  from, 

42-47 

Nuttall's  method,  43 
small  amounts  of  blood  from,  41 
serum,   normal,   non-specific   comple- 
ment-fixation by,  419 
Rabies,  681 
diagnosis,  683 
incubation  period,  683 
management,  683 
nature,  682 
Negri  bodies,  682 
Pasteur  treatment,  intensive.  686 
mild,  687 
principle,  685 
results,  687 


Rabies  vaccine,  preparation,  685,  686 

virus  fixe  of,  685 
Rachicentesis,   37.        See  also   Lumbar 

puncture. 
Racial  immunity,  168 

susceptibility,  101 
Rashes  in  serum  disease,  610-612 
multiform,  611 
scarlatiniform,  612 
urticarial,  611 
Rats,  intravenous  inoculation,  60 

obtaining  large  amount  of  blood  from, 

47 

small  amount  of  blood  from,  47 
white,  anaphylaxis  in,  576 
Reaction,  Abderhalden's  pregnancy,  265. 
See  also  Abderhalden's  serodiagnosis 
of  pregnancy. 
abortin,  648 

agglutination,  294.     See  also  Aggluti- 
nation test. 
allergic,  skin,  620.    .See  Anaphylactic 

reaction. 
anaphylactic,  620.      See  Anaphylactic 

reaction. 

antilysin,   for  antistaphylococcus   se- 
rum, 249 
antitrypsin,  264 

Bergmannn  and  Meyer's,  264 
Ascoli  and    Izar's    miostagmin,   563. 

See  also  Miostagmin  reaction. 
bacteriolytic,  364.    See  also  Bacterio- 

lytic  test. 
Bauer's  modification  of  Wassermann, 

482 
Bergmann  and   Meyer's  antitrypsin, 

264 

biologic  blood,  for  detection  of  blood- 
stains, 326 
technic,  331 
of  meat  adulteration,  333 

technic,  335 

blood,  biologic,  for  detection  of  blood- 
stains, 326 
technic,  331 
of  meat  adulteration,  333 

technic,  335 
Teichmann's,  326 
body,  588 

Bordet-Gengou,  354,  413.  See  also 
Bordet-Gengou  phenomenon  of  com- 
plement fixation. 

Browning  and  Mackenzie's  modifica- 
tion of  Wassermann,  484 
Calmette  tuberculin,  628,  639 
dangers,  633 
in  animals,  641 
Castellani's  saturation,  309 
cobra  venom,  405.      See  also  Venom 

hemolysis. 

complement-fixation,  354,  413.       See 
also   Bordet-Gengou   phenomenon   of 
complement  fixation. 
Craig's  modification  of  Wassermann, 
482 


INDEX 


967 


Reaction,  cutaneous,  in  typhoid  fever, 
650 

cytotoxic,  541 
in  diagnosis,  541 

of  Freund  and  Kaminer,  for  cancer, 
541 

Detre  and  Brezovsky's  modification 
of  Wassermann,  484 

epiphanin,  559.  See  also  Epiphanin 
reaction. 

for  seminal  stain,  327 

Fornet's  ring,  for  syphilis,  321 

Freund  and  Kaminer 's  cytolytic  can- 
cer, 541 

gonococcus  complement-fixation,  503. 
See  also  Gonococcus  complement-fixa- 
tion test. 

Gruber-Widal,  in  typhoid  fever,  281, 
290,  295,  299 

Hecht-Weinberg-Gradwohl  modifica- 
of  Wassermann,  482 

Herman-Perutz,  for  syphilis,  322 

immunity,  606 

Jarisch-Herxheimer,  874 

Koch's  tuberculin,  628,  633 
in  animals,  641 

Kolle  and  Pfeiffer's  macroscopic  ag- 
glutination, 305 

Lange's  colloidal  gold,  555 

luetin,  642.     See  also  Luetin  reaction. 

mallein,  647.  See  also  Mallein  reac- 
tion. 

Mantoux  tuberculin,  628,  636 

McDonagh  gel  for  syphilis,  323 

Mendel  tuberculin,  628,  636 
in  animals,  642 

miostagmin,  563.  See  also  Miostag- 
min  reaction. 

Moro  tuberculin,  628,  640 

Much's  psycho-,  410 

Noguchi's   butyric-acid,    for   protein, 

322 

luetin,  642.      See  also  Luetin  reac- 
tion. 

modification  of  Wassermann,  475. 
See  also  Noguchi's  modification. 

ophthalmic,  in  typhoid  fever,  649 

Pfeiffer's  bacteriolytic,  364 
experimental  work,  938 
in  diagnosis  of  disease,  370 
technic,  369 

precipitin.     See  Precipitin  test. 

psycho-,  of  Much,  410 

Purges-Meier,  for  syphilis,  321 

saturation,  of  Castellani,  309 

Schick,  for  natural  diphtheria  anti- 
toxin, 228 

Seifert's  epiphanin,  562 

Stern's  modification  of  Wassermann, 
484 

Tchernogubou's  modification  of  Was- 
sermann, 484 

Teichmann's  blood,  326 

toxin-antitoxin,  nature  of  experimental 
reaction,  912 


Reaction,  tuberculin,  623.     See  also  Tu- 
berculin reaction. 
typhoidin,  650 
vaccinoid,  606 

modification  of  Wassermann,  485 
von  Pirquet's  tuberculin,  628,  637 

in  animals,  642 
Wassermann,  in  syphilis,  425.   See  also 

Wassermann  reaction. 
Weichardt's  epiphanin,  559.     See  also 

Epiphanin  reaction. 
Widal,  in  typhoid  fever,  experimental 

work,  916 

Wolff-Eisner  tuberculin,  628,  639 
dangers,  633 
in  animals,  641 
Reagin,  syphilis,  429 
Receptoric  atrophy,  80,  173 
Receptors,  149 

of  first  order,  151,  227 
of  third  order,  339 
three  orders,  151 
Rectal  injections  of  serum  as  preventive 

of  serum  disease,  613 
method   of   administering   diphtheria 

antitoxin,  759 

Relapsing  fever,  salvarsan  in,  881 
Wassermann  reaction  in,  491 
Remittent  infection,  138 
Resistance,  drug,  849 
Resistant  races,  850 
Respiratory  diseases,   vaccine  therapy, 

708 

organs  as  portal  of  entrance  for  bac- 
teria, 88 

Retention  theory  of  immunity,  146 
Revaccination,  smallpox,  679 
Rhinitis,  vaccine  therapy,  708 
Richet's  studies  on  anaphylaxis,  569 

theory  of  anaphylaxis,  591 
Ricin  toxin,  120 

Ring  test,  Fornet's,  for  syphilis,  321 
Ringworm,  vaccine  therapy,  706 
Rubber  teats  for  capillary  pipets,  20 
Ruck's  tuberculin,  preparation  of,  718 


SALT  solution,  27 

resistance   of   erythrocytes   to,    ex- 
perimental work,  923 
Salvarsan,  845 

experimental  work,  944 

in  acanthosis  nigricans,  881 

in  Aleppo  boil,  881 

in  anemia,  881 

in  chancroid,  881 

in  chorea,  881 

in  concentrated  solution,  intravenous 
administration,  865 

in  dermatitis  herpetiformis,  881 

in  Duhring's  disease,  881 

in  filariasis,  881 

in  frambesia,  880 

in  Hodgkin's  disease,  881 

in  internal  anthrax,  821 


968 


INDEX 


Salvarsan  in  keratosis  follicularis,  881 
in  leprosy,  881 
in  lichen  planus,  881 
in  lupus  vulgaris,  881 
in  malaria,  881 
in  mycosis  fungoides,  881 
in  non-syphilitic  diseases,  880 
in  pellagra,  881 
in  pityriasis  rubra,  881 
in  psoriasis,  881 
in  relapsing  fever,  881 
in  scarlet  fever,  881 
in  scurvy,  881 
in  smallpox,  881 
in  syphilis,  852 
acid  solution,  857 
administration,  858 
alkaline  solution  of  disodium  salt, 

858 

contraindications,  878 
effect  of,  on  Wassermann  reaction, 

495 

history,  852 

intramuscular  injection,  875 
intravenous  injection,   858.        See 
also  Intravenous  injection  of  sal- 
varsan. 

methods  of  preparing,  for  adminis- 
tration, 857 

mono-acid  solution,  857 
neutral  suspension,  858 
precautions,  865 
value,  879 
in  trichinosis,  881 
in  tropical  ulcer,  881 
in  trypanosomiasis,  881 
in  tuberculosis,  881 
in  verruca  plana,  881 
in  Vincent's  angina,  881 
in  yaws,  880 

preparation  of  dilute  solution  for  in- 
travenous injection,  860 
properties  of,  854 

reactive  manifestations  after  use,  867 
causes  of  reaction,  869 
early  symptoms,  868 
immediate  symptoms,  868 
late  symptoms,  869 
sodium,  856 

studies  in  toxicity  of,  855 
Salvarsanized  autoserum  in  syphilis  of 
brain   and   spinal   cord, 
836 

after-treatment,  839 
repeating  dose,  840 
serobiologic     findings     in 
cerebrospinal  fluid,  840 
technic,  838 
serum,  intraspinous  injection,  876 

Fordyce's  technic,  877 
Salvarsannatrium,  856 
Saponin,  554   - 
Sapremia,  93 
Saprophytes,  83,  125 
Sarcoma,  epiphanin  reaction  in,  563 


Saturation   agglutination   reaction,    ex- 
perimental work,  919 
test  of  Castellani,  309 
Scar  in  smallpox  vaccination,  678 
Scarlatiniform  rashes  in  serum  disease, 

612 
Scarlet   fever,    antistreptococcus   serum 

in,  817 

salvarsan  in,  881 

treatment   with   specific   serum   of 
convalescents   and    with   normal 
serum,  826 
vaccination,  699 
Wassermann  reaction  in,  491 
Schick  test  for  natural  diphtheria  anti- 
toxin, 228,  771 

in  hospitals  and  institutions,  232 
intracutaneous  injection,  230 
intradermal  injection,  229 
method  of  applying,  230 

of  preparing  toxin,  230 
practical  value,  231,  232 
principles,  228 
reading  reaction,  231 
reactions  at  various  ages,  232 
Sclavo's  serum,  820 
Scurvy,  salvarsan  in,  881 
Seifert's  epiphanin  reaction,  562 
Selenium-eosin  compound  in  cancer,  888 
Seminal  stains,  test  for,  327 
Sensibilisin,  588,  592 
Sensibilisinogen,  592 
Sensitization,  572 
Sensitized  vaccines,  665 

bacterial,  preparation  of,  220 
Sensitizers,  344 
Sensitizing  substance,  148 
Sepsis,  puerperal,  antistreptococcus  se- 
rum in,  817 
vaccine  therapy,  713 
Septicemia,  94 
Serobacterin,  665 

Serodiagnosis  .of  pregnancy,   Abderhal- 
den's,  265.       See  also  Abderhalden' s 
serodiagnosis  of  pregnancy. 
Sero-enzymes  in  acute  infections,  278 
in  cancer,  277 
in  disease,  277 
in  mental  diseases,  278 
in  syphilis,  278 
in  tuberculosis,  278 

Serous  membranes,  tuberculosis  of,  auto- 
serum  treatment,  841 
tuberculin   test   in   diagnosis   of, 

632 

Serum  and  food  hypersensitiveness,  al- 
lergic reactions  in,  652 
active,  350 
agglutinating,  68 

power,  variation  in,  288 
amboceptor  unit,  396 
and  corpuscles,  obtaining  of,  30 
anti-anthrax,  820 

administration  of,  820 
anticholera,  823 


INDEX 


969 


Serum    anticomplementary   action,    ex- 
perimental work,  933 
anticytotoxic,  538 
antidiphtheric,  production  of,  235 
antidysenteric,  collecting  and  testing, 
248 

Flexner    and    Amoss    method    for 
rapid  production  of,  247 

production  of,  245 
culture,  246 

immunizing  animals,  247 
antigonococcus,  818 

action  of,  819 

administration  of,  819 

preparation  of,  819 
anti-influenza,  803 

administration  of,  804 
antimeningococcus,    788.         See  also 

Antimeningococcus  serum. 
antiplague,  822 
antipneumococcus,    810.        See    also 

Antipneumococcus  serum. 
antistaphylococcus,  249,  819 

antilysin  test  for,  249 

preparation  of,  249 

antistreptococcus,  813.    See  also  Anti- 
streptococcus  serum. 
antitetanic,  production  of,  243 
antitryptic    power,    testing,    experi- 
mental work,  914 
antituberculosis,  824 
antityphoid,  821 
auto-,  treatment  with,  835.     See  also 

Autoserum  treatment. 
bacteriolytic,  in  treatment  of  disease, 
381 

method  of  titrating,  366 

production  of,  69 
cadaver,   testing  of,   in  .  Wassennann 

reaction,  435 
calf  cholera,  785 
Chantemesse's,  821 
cytotoxic,  production  of,  73 
diagnosis  of  paroxysmal  hemoglobin- 

uria,  401 
disease,  570,  573,  608 

accelerated  reaction  in,  610 

atropin  sulphate  as  preventive,  613 

detection  of,  612 

from  antimeningococcus  serum,  799 

from  diphtheria  antitoxin,  764 

immediate  reaction  in,  610 

multiform  rashes  in,  611 

nature,  609 

prevention,  612 

rashes  in,  610-612 

rectal  injections  of  serum  as  pre- 
ventive, 613 

scarlatiniform  rashes  in,  612 

severe,  612 

symptoms,  610 

treatment,  614 

urticarial  rashes  in,  611 
doses  of,  in  Wassermann  reaction,  435 
hemolysis,  experimental  work,  924 


Serum  hemolysis,  quantitative   factors, 

experimental  work,  925. 
hemolytic,  production  of,  71 
hog  cholera,  production  of,  784 

standardization  of,  784 
immune,  and  normal  serum,  difference 

between,  346 
methods  for  making,  66 
preservation  of,  76 

in  dried  paper  form,  80 

in   fluid   form,    by   bacteria-free 

filtration,  76 
by  freezing,  78 
with  antiseptics,  76 
in  living  animal,  80 
in  powder  form,  79 
standardization     of,     complement- 
fixation  test  in,  524 
inactivated,  349,  350 
intramuscular  inoculation,  technic,  742 
intravenous       inoculation,       gravity 

method,  743 
syringe  method,  742 
technic,  742 
Kraus'  anticholera,  823 
Maragliano's  tuberculosis,  824 
Marmorek's  tuberculosis,  824  • 
methods  of  inoculation  with,  739 
nephrotoxic,  production  of,  73 
normal,  829.       See  also  Serum  treat- 
ment, normal. 

and  immune  serum,  difference  be- 
tween, 346 
preservation  of,  75 
obtaining  of,  30 

patient's  own,  835.       See  also  Auto- 
serum  treatment. 

placental,  method  of  obtaining,  833 
precipitins,  70 

experimental  work,  920 
production  of,  70 
preservation  of,  75 
reactivated,  350 
rectal    injections,    as    preventive    of 

serum  disease,  613 
salvarsanized,   intraspinous  injection, 

876 

Fordyce's  technic,  877 
Sclavo's,  820 

sickness,  570,  573,  608.      See  also  Se- 
rum disease. 

subcutaneous  inoculation,  technic,  740 
subdural    inoculation,    technic,    746. 

See  also  Subdural  inoculation. 
treatment,  657,  733 
anaphylaxis  in,  738 
asthma  in,  739 
contraindications  to,  738 
indications,  736 
intramuscular  inoculation,  technic, 

742 
intravenous     inoculation,     gravity 

method,  743 
syringe  method,  742 
technic,  742 


970 


INDEX 


Serum  treatment,  methods  of  inocula- 
tion, 739 
normal,  829 

in  hemorrhage,  829 
in  nephritis,  834 
in  skin  diseases,  834 
in  toxicoses  of  pregnancy,  833 
in  vomiting  of  pregnancy,  833 
of  acute  anterior  poliomyelitis,  826 
of  anthrax,  820 
of  bubonic  plague,  822 
of  cerebrospinal  meningitis,  788 
of  cholera,  823 

of  diphtheria,  754.      See  also  Diph- 
theria antitoxin. 
of  dysentery,  782 

results,  783 

of  gonococcal  infections,  818 
of  hay-fever,  786 
of  hog  cholera,  784 
of  infantile  paralysis,  826 
of  infectious  diseases  with  specific 
serum  of  convalescents  and  with 
normal  serum,  825 
of  influenzal  meningitis,  802 
of  internal  anthrax,  821 
of  meningococcus  meningitis,  788 
cases  of  posterior  basal  menin- 
gitis, 798 

with  dry  canal,  798 
with  thick  elastic  exudate,  798 
chronic  cases,  799 
results,  799 
serum  sickness  in,  799 
subacute  cases,  799 
of  plague,  822 
of  pneumococcus  infections,  807 

meningitis,  804,  805. 
of  pneumonia,  807,  828 

results,  812 
of  scarlet  fever,  825 
of  snake-bites,  786 
of  staphylococcus  infections,  819 
of  streptococcus  infections,  813 
of  tetanus,  772.      See  also  Tetanus 

antitoxin. 

of  tuberculosis,  824 
of  typhoid  fever,  821 
purposes,  734 
status  lymphaticus  in,  739 
subcutaneous  inoculation,   technic, 

740 
subdural  inoculation,  technic,  746. 

See  also  Subdural  inoculation. 
Sheep,  anaphylaxis  in,  577 
intravenous  inoculation,  62 
obtaining  large  amount  of  blood  from, 

48 

small  amount  of  blood  from,  42,  49 
Side-chain  theory  of  immunity,  148 

and  phagocytic  theory,  compati- 
bility, 157 

Simon    and    Lamar's    modification    of 

Wright's  method  for  opsonic  index,  203 

Skin  as  portal  of  entrance  for  bacteria,  87 


Skin,  bacteria  on,  85,  87 

diseases,  autoserum  treatment,  835 
normal  serum  treatment,  834 
salvarsan  in,  881 
vaccine  therapy,  704 
reactions,  allergic,  620 

as  indices  of  immunity,  622 

of  infection  and  hypersuscepti- 

bility,  621 
effect  of  drugs,  644 
etiology,  620 

tuberculosis  of,  tuberculin  test  in  diag- 
nosis of,  631 
treatment,  731 
Smallpox,    antistreptococcus   serum   in, 

817 

revaccination,  679 
salvarsan  in,  881 
vaccination,  668.  See  also  Vaccination, 

smallpox. 

vaccinia  and,  relationship,  670 
Smith's  phenomenon  of  anaphylaxis,  571 
Snake  venoms,  121,  251 
nature  of,  121 
properties  of,  121 
Snake-bites,  serum  treatment,  786 
Sodium  citrate,  27 

salvarsan,  856 
Sol,  definition,  544 
Solutions,  27 

coUoidal,  544 

Southard  and  Gay's  theory  of  anaphy- 
laxis, 592 
Species  immunity,  168 

susceptibility,  101 
Spermatotoxin,  538 
Spinal  cord,  syphilis  of,  salvarsanized 

autoserum  in,  836 
after-treatment,  839 
repeating  dose,  840 
serobiologic  findings  in  cere- 
brospinal fluid,  840 
technic,  838 

puncture,  37.     See  also  Lumbar  punc- 
ture. 

Splitting  of  complement,  353 
Spontaneous  phagocytosis,  190 
Sporadic  infection,  82 
Sporotrichosis,  allergic  reactions  in,  653 
Stain,  blood,  biologic  test  for,  326 

technic,  331 
experimental  work,  921 
identification   of,   complement-fixa- 
tion test  for,  527 
test  for,  experimental  work,  937 
seminal,  test  for,  327 
Staining  acid-fast  bacilli,  200 

bacteria,  200 

Stalagmometer,  Traube's,  563 
Standardization    of    antimeningococcus 

serum,  792 

of  antipneumococcus  serum,  811 
of  antistreptococcus  serum,  816 
of  bacterial  antigens  in  complement 
fixation,  500 


INDEX 


971 


Standardization  of    bacterial   vaccines, 

214 

of  diphtheria  antitoxin,  240 
experimental  work,  911 
toxin,  115 

of  hog  cholera  serum,  784 
of  immune  serums,  complement-fixa- 
tion test  in,  524 
of  tetanus  antitoxin,  244,  772 

experimental  work,  912 
Staphylococcus,  118 

infections,  serum  treatment,  819 
vaccine,     preparation,     experimental 

work,  911 
Staphylolysin,  118 

method  of  titrating,  250 
preparation  of,  250 

Staphylotoxin,  experimental  work,  897 
Status  lymphaticus  in  serum  treatment, 

739 
Sterilization  of  bacterial  vaccines,  217 

testing,  217 
of  pipets,  22 
of  syringes,  27 
of  test-tubes,  25 

Stern  and  Korte's  method  of  measuring 
bactericidal  power  of 
blood,  371 

experimental  work,  939 
Stern's  modification  of  Wassermann  re- 
action, 484 
Stichreaktion,  627 

Stimulation,    provocatory,    of    Wasser- 
mann reaction,  495 
Stimulins,  147,  190 
Stock  bacterial  vaccines,  665 
Strawberries,  idiosyncrasy,  615 
Streptococcus,  119 

infections,  serum  treatment,  813 
Streptolysin,  119 

Streptotoxin,  experimental  work,  898 
Strong  and  Kolle's  plague  vaccine,  695 
Strong's  cholera  vaccine,  preparation,  698 
Subcutaneous  injection  of  animals,  54 
with  fluid  inoculum,  54 
with  solid  inoculum,  55 
of  diphtheria  antitoxin,  759 
of  serum,  technic,  740 
of  tetanus  antitoxin,  774 
of  tuberculin,  723,  727 
mallein  reaction,  648 
tuberculin  reaction,  628,  633 

in  animals,  641 
Subdural  injection  of  antimeningococcus 

serum,  795 
of  neosalvarsan  in  syphilis,  875 

Wile's  technic,  876 
of  salvarsanized  serum,  876 
of  serum,  anesthesia  for,  749 
blood-pressure  as  guide,  747 
collapse  in,  746,  748 
symptoms,  748 
treatment,  748 
gravity  method,  749 
syringe  method,  752 


Subdural  injection   of    serum,   technic, 

746 

of  tetanus  antitoxin,  775 
Subinfection,  81,  89 
Substance  sensibilisatrice,  187,  341,  344, 

359,  384 

Suction  pump,  29 

Suctorial  insects,   transmission  of  bac- 
teria by,  86 

Susceptibility,  acquired,  101 
familial,  101 

general,  in  relation  to  infection,  100 
individual,  101 
inherited,  101 
racial,  101 
species,  101 
Suspensions,  544 

colloidal,  544 

Sycosis,  vaccine  therapy,  704 
Synocytotoxin,  539 

Syphilis  after  smallpox  vaccination,  680 
agglutination  in,  292 
Bauer's  modification  of  Wassermann 

reaction,  482 

Browning  and  Mackenzie's  modifica- 
tion of  Wassermann  reaction,  484 
cerebrospinal,  Lange's  colloidal   gold 

test  in,  559 
complement  fixation  in,  425.    See  also 

Wassermann  reaction. 
Craig's  modification  of  Wassermann 

reaction,  482 
Detre  and   Brezovsky's   modification 

of  Wassermann  reaction,  484 
epiphanin  reaction  in,  563 
Fornet's  ring  test  for,  321 
Hecht-Weinberg-Gradwohl    modifica- 
tion of  Wassermann  reaction,  482 
Herman-Perutz  test  for,  322 
horse,  complement-fixation  test  in,  515 
luetin  reaction  for,  642.        See  also 

Luetin  reaction. 
McDonagh  gel  test,  323 
mercury  treatment,  effect  of,  on  Was- 
sermann reaction,  492 
miostagmin  reaction  in,  566 
neosalvarsan  in,  852.      See  also  Neo- 
salvarsan in  syphilis. 
Noguchi's    modification    of    Wasser- 
mann reaction,  475.      See  also  No- 
guchi's  modification. 
of  brain,  salvarsanized  autoserum  in, 

836 

after-treatment,  839 
repeating  dose,  840 
serobiologic    findings    in    cere- 
brospinal fluid,  840 
technic,  838 

of    spinal    cord,    salvarsanized    auto- 
serum,  836 
after-treatment,  839 
repeating  dose,  840 
serobiologic  findings  in  cere- 
brospinal fluid,  840 
technic,  838 


972 


INDEX 


Syphilis,  Forges-Meier  test  for,  321 
reagin,  429 
salvarsan  in,  852.     See  also  Salvarsan 

in  syphilis. 
sero-enzymes  in,  278 
Stern's  modification  of  Wassermann 

reaction,  484 

Tchernogubou's  modification  of  Was- 
sermann reaction,  484 
venom  hemolysis  in,  407 

experimental  work,  938 
practical  value,  410 
technic,  407 

von  Dungern's  modification  of  Was- 
sermann reaction,  485 
Wassermann  reaction  in,  425.       See 

also  Wassermann  reaction. 
Syphilitic  livers,  alcoholic  extracts,  for 

Wassermann  reaction,  444 
aqueous  extracts,  for  Wassermann 

reaction,  443 
Syringe,  26 

for  administration  of  bacterial  vac- 
cines, 221 
Kitchens',  241 
method  of  intravenous  inoculation  of 

serum,  742 
of  subdural  inoculation  of  serum, 

752 

sterilization  of,  27 
Systemic  reaction  to  infection,  139 


TABES  dorsalis,  Lange's   colloidal   gold 

test  in,  559 

Wassermann  reaction  in,  488 
T.  B.  tuberculin,  718 
Tchernogubou's  modification  of  Wasser- 
mann reaction,  484 

Teats,  rubber,  for  capillary  pipets,  20 
Technic,  general,  17 
Teichmann's  blood  test,  326 
Temperature,   influence  of,   on  natural 

immunity,  experimental  work,  903 
Terni-Bandi's     method     of     preparing 

plague  serum,  823 
plague  vaccine,  696 
Tertiary  syphilis,  Wassermann  reaction 

in,  487 

Test.    See  Reaction. 
Test-tubes,  conversion  of,  into  ampules 

for  holding  vaccine,  etc.,  24 
for  immunologic  work,  25 
for  Wassermann  reaction,  431 
sterilization  of,  25 
Tetanolysin,  113,  117,  895 
Tetanospasmin,  113,  117,  895 
Tetanus  after  smallpox  vaccination,  679 
antitoxin,  772,  778 
action,  773 

intramuscular  injection,  774 
intraneural  injection,  774 
intravenous  injection,  775 
method  of  titrating,  245 
methods  of  administering,  774 


Tetanus  antitoxin,  preparation,  772 
production  of,  243 
collecting  serum,  244 
immunizing  animals,  244 
prophylactic  administration,    775, 

777 

results  of  treatment,  780 
standardization,  244,  772 
experimental  work,  912 
subcutaneous  injection,  774 
subdural  injection,  775 
unit  of,  252 
bacillus,  117 
prophylaxis,  775 
serum    treatment,    772.        See    also 

Tetanus  antitoxin. 
surgical  treatment,  777,  778 
toxin,  117 
action,  772 

experimental  work,  895 
production  of,  243 
treatment  of,   772,   778.        See  also 

Tetanus  antitoxin. 
Therapeutic  immunization,  618 
Therapia  magna  sterilisans,  851 
Thyrotoxins,  540 
Tissue  affinity,  selective,  99 

of  Treponema  pallidum  strains,  99 
concerned  in  production  of  antibodies, 

164 
susceptibility,  avenue  of  infection  and, 

98 

T.  O.  tuberculin,  717 
Tonsillar  diphtheria,  dosage  of  antitoxin 

in,  762 
Topfer  and  Jaffe's  method  of  measuring 

bactericidal  power  of  blood,  374 
Toxemia,  94 
Toxicity  of  bacteria,  95 
Toxicoses  of  pregnancy,  normal  serum 

treatment,  833 
Toxin,  73,  109 
abrin,  120 

and  ferment,  similarity  between,  254 
botulism,  118 

experimental  work,  896 
crotin,  120 
diphtheria,  113 

experimental  work,  893 
limes  death  dose,  240 

zero  dose,  241 
production  of,  235 
reaction  in  horses,  238 
standardizing,  115 
testing,  236 

virulence,  114 
dysentery,  118 

experimental  work,  896 
exogenous,  109 
extracellular,  109,  110 
chemical  properties,  111 
general  properties,  110 
precipitation  of,  111 
haptophore  group,  112 
nature  of,  112 


INDEX 


973 


Toxin,  nomenclature,  109 
of  hay-fever,  120 
of  higher  plants  and  animals,  119 
pollen,  120 

principal,  special  properties,  113 
ricin,  120 

selective  action,  112 
soluble,  109 

chemical  properties,  111 
general  properties,  110 
structure  of,  111 
tetanus,  117 
action  of,  772 
experimental  work,  895 
production  of,  243 
toxophore  group,  112 
Toxin-antitoxin  reaction,  nature  of,  232 

experimental  work,  912 
Toxogen,  588 
Toxogenin,  591 
Toxoid,  112 

diphtheria,  117 
Toxon,  113 

Toxophore  group  of  toxin,  112 
T.  R.  tuberculin,  dose  of,  726 

preparation  of,  717 
Transfusion,  blood,  287 

experimental  work,  920 

methods,  831 

relation  of  isohemagglutinins  to,  286 

technic,  832 

tests  before,  for  isohemagglutinins 

and  isohemolysins,  311 
Brem's  microscopic  hemag- 
glutination  method,  312 
Traube's  stalagmometer,  563 
Trauma  as  predisposing  to  infection,  103 
Treatment,  cytotoxins  in,  540 

effect  of,  on  Wassermann  reaction,  492 
Treponema  pallidum,  agglutination  of, 

292 

aqueous  extract  of  culture,  for  Was- 
sermann reaction,  448 
selective  tissue  affinity  of  strains,  99 
Trichina  spiralis,  169 
Trichinosis,  salvarsan  in,  881 
Tritotoxin,  116 

Tropical  ulcer,  salvarsan  in,  881 
Trypanosomiasis,  salvarsan  in,  881 
Tubercle  bacilli,  living,  in  treatment  of 

tuberculosis,  721 
Tuberculin,  action  of,  720 

administration  of,  methods,  723,  728 
bacillen  emulsion,  preparation  of,  718 
B.  E.,  preparation  of,  718 
Beraneck's,  dose  of,  727 

preparation  of,  718 
B.  F.,  preparation  of,  718 
bouillon  filtrate,  preparation  of,  718 
delirium,  724 
Dixon's,  dose  of,  727 
preparation  of,  719 
new,  dose  of,  726 

preparation  of,  717 
old,  dose  of,  726 


Tuberculin,  old,  preparation  of,  716 
O.  T.,  dose  of,  726 

preparation  of,  716 
preparation  of,  716 
reaction,  623 

Calmette's,  628,  639 
dangers,  633 
in  animals,  641 
comparative  delicacy  and  relation, 

628 

conjunctival,  of  Calmette,  628,  639 
dangers,  633 
in  animals,  641 
of  Wolff-Eisner,  628,  639 
dangers,  633 
in  animals,  641 
cutaneous,  of  von  Pirquet,  628,  637 

in  animals,  642 
dangers,  632 
delicacy,  628 
effect  of  measles  on,  627 
error  in,  sources,  626 
false  negative,  627 

positive,  626 
in  animals,  641 
in  diagnosis  of  pelvic  tuberculosis, 

631 

of  pulmonary  tuberculosis,  630 
of  tuberculosis  of  bones,  joints, 

and  glands,  631 
of  eye,  ear,  and  larynx,  631 
of  genito-urinary  system,  631 
of  serum  membrane,  632 
of  skin,  631 

of  tuberculous  meningitis,  632 
peritonitis,  632 
pleurisy,  632 
value,  630 

in  prognosis,  value,  632 
intracutaneous,   of    Mantoux,   628, 

636 
of  Mendel,  628,  636 

in  animals,  642 
Mantoux's,  628,  636 
Mendel's,  628,  636 

in  animals,  642 
methods  of  conducting,  628 
Moro's,  628,  640 
nature,  624 

percutaneous,  of  Moro,  628,  640 
relation,  628 
specificity,  624 
subcutaneous,  628,  633 

in  animals,  641 
von  Pirquet's,  628,  637 

in  animals,  642 
Wolff-Eisner's,  628,  639 
dangers,  633 
in  animals,  641 
residue,  preparation,  717 
subcutaneous  injection,  723 
T.  O.,  717 
T.  R.,  dose  of,  726 

preparation  of,  717 
treatment,  713 


974 


INDEX 


Tuberculin  treatment,  action  of  tuber- 
culin, 720 

contraindications,  723 
dosage,  726 
history,  713 
in  tuberculosis  of  bones  and  joints, 

731 

of  ear,  731 
of  eye,  731 

of  genito-urinary  organs,  732 
of  intestine,  731 
of  mesenteric  glands,  731 
of  skin,  731 
in  tuberculous  adenitis,  731 

meningitis,  731 
intrabronchially,  729 
intrafocal  route,  729 
intravenous,  729 
living  tubercle  bacilli,  721 
methods  of  administration,  723,  728 
oral  route,  728 
patients  suitable,  722 
preparation  of  tuberculin,  716 
reaction  in,  724 

constitutional  symptoms,  724 
focal  signs,  725 
local  signs,  725 
results,  730 

subcutaneous  injection,  723,  727 
von  Ruck's,  preparation  of,  718 
Tuberculonastin,  162 
Tuberculosis,  agglutination  test  in,  292 
antistreptococcus  serum  in,  817 
chemotherapy  in,  887 
complement-fixation  test  in,  517 
contraindications  to  vaccines  in,  667 
Friedmann's  treatment,  722 
genito-urinary,  tuberculin  test  in  diag- 
nosis of,  631 
living  tubercle  bacilli  in  treatment  of, 

721 

miostagmin  reaction  in,  566 
of  bones  and  joints,  tuberculin  treat- 
ment, 731 

tuberculin  test  in  diagnosis  of,  631 
of  ear,  tuberculin  test  in  diagnosis  of, 

631 

treatment,  731 
of  eye,  tuberculin  test  in  diagnosis  of, 

631 

treatment,  731 
of   genito-urinary   organs,   tuberculin 

treatment,  732 
of  glands,  tuberculin  test  in  diagnosis 

of,  631 

of  intestine,  tuberculin  treatment,  731 
of  joints,  tuberculin  test  in  diagnosis 

of,  631 
of  larynx,  tuberculin  test  in  diagnosis 

of,  631 

of  mesenteric  glands,  tuberculin  treat- 
ment, 731 

of  serous  membranes,  autoserum  treat- 
ment, 841 
tuberculin  test  in  diagnosis  of,  632 


Tuberculosis  of  skin,  tuberculin  test  in 

diagnosis  of,  631 
treatment,  731 
pelvic,  tuberculin  test  in  diagnosis  of, 

631 

pulmonary,  tuberculin  reaction  in  di- 
agnosis of,  730 
salvarsan  in,  881 
sero-enzymes  in,  278 
serum  treatment,  824 
tuberculin  treatment,  713.      See  also 

Tuberculin  treatment. 
venom  hemolysis  in,  412 
Tuberculous  adenitis,  tuberculin  treat- 
ment, 731 

meningitis,  autoserum  treatment,  843 
tuberculin  test  in  diagnosis  of,  632 

treatment,  731 
peritonitis,  tuberculin  test  in  diagnosis 

of,  632 

pleurisy,  autoserum  treatment,  841 
tuberculin  test  in  diagnosis  of,  632 
Typhoid  fever,  agglutination  test  in,  290, 

294,  299 

allergic  reactions  in,  649 
autoserum  treatment,  836 
complement-fixation  test  in,  516 
cutaneous  reaction  in,  650 
Gruber-Widal  reaction  in,  281,  290, 

295,  299 

miostagmin  reaction  in,  566 
ophthalmic  reaction  in,  650 
Oxford  University  agglutination  test 

in,  305 

serum  treatment,  821 
vaccination,  688 

dosage  of  vaccine,  690 
duration  and  degree,  691 
method  of  inoculation,  689 
preparation  of  vaccine,  688 
reactions,  690 
recommendations,  692 
results,  692 
vaccine  therapy,  711 
Widal    reaction    in,     experimental 

work,  916 
vaccine,     preparation,     experimental 

work,  910 

sensitized,  of  Gay  and  Claypool,  691 
Typhoidin  of  Gay  and  Claypool,  650 

reaction,  650,  651 
Typho-protein,  649 

Typhus  fever,  prophylactic  immuniza- 
tion in,  694 


UFFENHEIMER'S  method  of  determining 
presence  of  diphtheria  toxin,  116 

Uhlenhuth  filter,  328 

Ulcer,  tropical,  salvarsan  in,  881 

Ulcerative  endocarditis,  vaccine  therapv, 
713 

Umstimmung,  642 

Unit,  amboceptor,  of  serum,  396 
antitoxin,  252 


INDEX 


975 


Unit  of  diphtheria  antitoxin,  253 

of  tetanus  antitoxin,  253 
Urethritis,  vaccine  therapy,  707 
Urticarial  rashes  in  serum  disease,  611 

VACCINATION,    65,    654,  656,  657,  668, 

734 

active  acquired  immunity  by,  173 
anthrax,  700 
blackleg,  702 
bubonic  plague,  694 
cerebrospinal  meningitis,  699 
cholera,  697 

dosage  of  vaccine,  698 
Haffkine's  vaccine,  697 
Kolle's  vaccine,  697 
preparation  of  vaccine,  697 
results  of,  698 
Strong's  vaccine,  698 
diphtheria,  767 
dysentery,  699 
history  of,  654 
Jennerian,  173 

meningococcus  meningitis,  801,  802 
of  animals,  671 

preparation  for,  672 
paratyphoid  fever,  693 
Pasteurian,  173 
pertussis,  700 
plague,  694 

dosage  of  vaccine,  696 
duration  of,  696 
effects  of,  696 
Haffkine's  vaccine,  695 
dosage,  696 
effects,  696 
results,  696 

Kolle  and  Strong's  vaccine,  695 
Kolle's  vaccine,  695 
Lustig  and  Galeotti's  vaccine,  695 
preparation  of  vaccine,  695 
results  of,  696 

Terni  and  Bandi's  vaccine,  695 
pneumonia,  694 
preparation  of  animals  for,  672 
rabies,  681.   See  also  Rabies. 
scarlet  fever,  699 
smallpox,  668 
history  of,  668 
phenomena  of,  677 
preparation  of  vaccine,  670 
protective  value,  680 
revaccination,  679 
risks  of,  679 
scar  in,  678 

subsequent  care  of  wound,  677 
syphilis  after,  680 
technic  of,  675 
tetanus  after,  679 
typhoid  fever,  688.     See  also  Typhoid 

fever  vaccination. 
typhus  fever,  694 
Vaccine,  656 

ampules,  conversion  of  test-tubes  into, 
24 


Vaccine  ampules,  making  of,  24 
anthrax,  700 
autogenous,  665 
bacterial,  210,  656 
administration  of,  221 

frequency,  222 

syringe  for,  221 
autogenous,  665 
counting  of,  214 

Hopkins'  method,  216 

Kolle's  method,  216 

with    hemocytometer    chamber, 
215 

Wright's  method,  214 
definition  of,  210 
dosage  of,  222 

opsonic  index  as  guide  to,  208 
experimental  work,  910 
in  abscesses,  704 
in  acne,  704 
in  acute  infections,  713 
in  bacillary  dysentery,  713 
in  bronchitis,  710 
in  carbuncles,  704 
in  cystitis,  706 
in  dermatitis  venenata,  705 
in  ecthyma,  705 
in  eczema,  705 
in  erysipelas,  706 
in  focal  infections,  704 
in  furunculosis,  704 
in  genito-urinary  diseases,  706 
in  gonorrheal  arthritis,  22,  707 
in  hay-fever,  708 
in  impetigo,  705 
in  infected  wounds,  703 
in  otitis  media,  711 
in  pertussis,  710 
in  pneumonia,  712 
in  puerperal  sepsis,  713 
in  pyelitis,  706 
in  respiratory  diseases,  708 
in  rhinitis,  708 
in  ringworm,  706 
in  skin  diseases,  704 
in  sycosis,  704 
in  typhoid  fever,  711 
in  ulceratiye  endocarditis,  713 
in  urethritis,  707 
in  vulvovaginitis  of  children,  708 
in  whooping-cough,  710 
inoculation  with,  dosage,  222 
effects  of,  222 
method  of  making,  221 
non-specific  activity,  663 
sensitized,  preparation  of,  220 
standardization  of,  214 
stock,  665 
technic  for  preparing,  210 

diluting  and   adding   preserva- 
tive, 218 
emulsion,  212 

procuring  infected  material,  210 
pure  cultures,  211 
sterilization,  217 


976 


INDEX 


Vaccine,  bacterial,  technic  for  preparing, 

sterilization,  testing,  217 
treatment  by,  702 
blackleg,  702 
cerebrospinal  699 
cholera,  preparation  of,  697 
contraindications  to,  667 
in  cancer,  668 
in  diabetes,  668 
in  nephritis,  668 

tuberculosis,  668 
cowpox,  preparation  of,  670 
animals,  671 
collection  of  virus,  672 
seed  virus,  671 
testing  virus,  674 
dead,  664 
dysentery,  699 

Haffkine's  cholera,  preparation,  697 
plague,  695 
dosage,  696 
effects,  696 
results,  696 

in  prophylaxis  of  disease,  654 
Kolle  and  Strong's  plague,  695 
Kolle's  cholera,  preparation,  697 

plague,  695 
living,  664 

Lustig  and  Galeptti's  plague,  695 
nomenclature,  656 
plague,  preparation  of,  695 
preparation  of,  method,  657 
rabies,  administration  of,  686 

preparation  of,  685 
scarlet  fever,  699 
sensitized,  665 
staphylococcus,    experimental    work, 

911 

stock,  665 

Strong's  cholera,  preparation,  698 
Terni  and  Bandi's  plague,  696 
treatment,  654,  657,  660 

history,  654 

typhoid,  experimental  work,  910 
preparation  of,  688 
sensitized,  of  Gay  and  Claypool,  691 
Vaccinia,  655,  656,  677 

smallpox  and,  relationship,  670 
Vaccinoid,  678 
reaction,  606 
Variola.     See  Smallpox. 
Varioloid,  679 
Vaughan  and  Wheeler  on  anaphylatoxin, 

58*4,  585 
and  Wheeler's  theory  of  anaphylaxis, 

592 
Vaughan's  theory  of  bacterial  proteins, 

130 

of  immunity,  175 
Vegetables,  idiosyncrasy,  615,  652 
Venesection,  33 

in  children,  33 
Venom,  cobra,  experimental  work,  899 

preparation  of,  407 
hemolysis,  405,  554 


Venom  hemolysis  in  cancer,  412 
in  syphilis,  407 

experimental  work,  938 
practical  value,  410 
technic,  407 
in  tuberculosis,  412 
nature,  405 
snake,  121,  251 
properties  of,  121 
nature  of,  121 

Verruca  plana,  salvarsan  in,  881 
Vincent's  angina,  salvarsan  in,  881 
Virulence  of  bacteria,  95 
decrease,  96 
increase,  96 

by  addition  of  animal  fluids  to 

culture-medium,  97 
by  passage  through  animals,  97 
by  use  of  collodion  sacs,  97 
Virus,    cowpox,   Noguchi's    method    of 

preparing,  674 
fixe  of  rabies,  145,  685 
Vomiting  of  pregnancy,  normal    serum 

treatment,  833 
von  Dungern's  modification  of  Wasser- 

mann  reaction,  485 
von  Pirquet's  cutaneous  tuberculin  test, 

628,  637 
in  animals,  642 
studies  on  anaphylaxis,  569 
Von  Ruck's  tuberculin,  preparation,  718 
Vulva,  diphtheria  of,  dosage  of  antitoxin 

in,  763 

Vulvovaginitis  of  children,  vaccine  ther- 
apy, 708 


WASHED  leukocytes  in  opsonic  index,  198 
Washing  erythrocytes,  28 
Wassermann  reaction  after  anesthesia, 

491 

Colics'  law  and,  489 
in  cerebral  syphilis,  488 
in  congenital  mental  deficiency,  490 

syphilis,  489,  490 
in  epilepsy,  490 
in  frambesia,  491 
in  general  paralysis,  487 
in  latent  syphilis,  488 
in  leprosy,  491 
in  malaria,  491 
in  paralytic  dementia,  487 
in  parasyphilitic  diseases,  487 
in  pellagra,  491 
in  primary  syphilis,  485 
in  relapsing  fever,  491 
in  scarlet  fever,  491 
in  secondary  syphilis,  486 
in  syphilis,  425 . 

acetone-insoluble  lipoids  for,  446 
alcoholic  extracts  of  normal  or- 
gans for,  444 
re-enforced  with  cholesterin 

for,  445 
of  syphilitic  livers  for,  444 


INDEX 


977 


Wassermann  reaction  in  syphilis,  anti- 
gen for,  441 
anticomplementary      titration, 

452 

antigenic  titration,  455 
experimental  work,  932 
hemolytic  titration,  454 
method  of  diluting,  451 

titrating,  452 
preparation,  443 

antigenic  dose  of  serum,  442 

antisheep  amboceptor  for,  439 

aqueous  extract  of  pallidum  cul- 
ture for,  448 
of  syphilitic  livers  for,  443 

as  colloidal  reaction,  552 

Bauer's  modification,  482 

Browning  and  Mackenzie's  modi- 
fication, 484 

cerebrospinal  fluid  for,  435 

collecting  blood  for,  432 

New  York  Board  of  Health 
outfit,  434 

Colics'  law  and,  489 

comparative  antigenic  values  of 
various  extracts,  448 

complement  for,  435 
preservation  of,  23,  437 

congenital,  489,  490 

Craig's  modification,  482 

Detre  and  Brezovsky's  modifica- 
tion, 484 

doses  of  serum  in,  435 

effect  of  mercury  treatment  on, 

492 

of  neosalvarsan  on,  495 
of  salvarsan  on,  495 
of  treatment  on,  492 

erythrocytes  for,  440 

experimental  work,  933,  934,  935 

first  method,  456 
technic,  459 

fluid  to  be  tested,  432 

fourth  method,  458,  470 
reading  results,  474 
technic,  470 

glassware  for,  431 

guinea-pig     complement     serum 
for,  436 

Hecht-Weinberg-Gradwohl  modi- 
fication, 482 

hemolytic  amboceptor,  for,  439 

history,  425 

latent,  488 

lecithin  and  cholesterin  for,  447 

methods  for  conducting,  4,56 

modifications,  475 

Noguchi's  modification,  475.    See 
also  Noguchi's  modification. 

original,  456,  459 
technic  of,  459 

pipets  for,  431 

practical  value,  495 

primary,  485 

principles  and  theories,  428 


Wassermann  reaction  in  syphilis,  Pro- 

f eta's  law  and,  490 
provocatory  stimulation,  495 
reading  and  recording,  463 
red  blood-corpuscles  for,  440 
relation  of  lipoids  to,  555 
second  method,  457,  465 
reading  results,  466 
technic,  465 
secondary,  486 
serum  for,  432 
specificity,  ,490 
Stern's  modification,  484 
Tchernogubou's  modification,  484 
technic,  431 
tertiary,  487 

testing  cadaver  serums,  435 
test-tubes  for,  431 
third  method,  458,  467 
reading  results,  469 
technic,  467 
various  stages,  485 
von  Dungern's  modification,  485 
with  multiple  antigens,  457 

technic,  465 

with  varying  amounts  of  comple- 
ment, 458 
technic,  470 
with  varying  amounts  of  patient's 

serum,  458 
technic,  467 
in  tabes  dorsalis,  488 
in  tertiary  syphilis,  487 
in  various  stages  of  syphilis,  485 
in  yaws,  491 
Profeta's  law  and,  490 
Water,  bacteria  in,  85 
Water-supplies,  bacteria  recovered  from, 
bacteriolytic  test  for  identification  of, 
365 

Wechsberg  and  Neisser's  method  of 
measuring  bactericidal  power  of  blood, 
371 

Wechselmann's    method    of    converting 
negative  syphilitic  serums  to  positive 
syphilitic  serums,  434 
Weichardt's    epiphanin    reaction,    559. 

See  also  Epiphanin  reaction. 
Weigert's  overproduction  theory,  150 
Welch,  hypothesis  of,  104 
Wet  cupping,  36 

Wheller  and  Vaughan's  theory  of  ana- 
phylaxis, 592 
White  mice,  anaphylaxis  in,  576 

rats,  anaphylaxis  in,  576 
Whooping-cough,  agglutination  reaction 

in,  293 

complement-fixation  in,  521 
prophylactic  immunization  in,  700 
vaccine  therapy,  710 
Widal  reaction  in  typhoid  fever,   281, 

290,  295,  299 
experimental  work,  916 
Wile's  technic  of  intraspinous  injection 
of  neosalvarsan,  876 


978 


INDEX 


Wolff-Eisner     conjunct!  val     tuberculin 

test,  628,  639 
dangers,  633 
in  animals,  641 

Wollstein  and  Amoss's  rapid  method  of 
production  of  antimeningococcus  se- 
rum, 792 

Wolman  and  Hamman's  method  of  pre- 
paring tuberculin  for  subcutane- 
ous tuberculin  test,  633 
plan  of  dosage  for  tuberculin  in  sub- 
cutaneous test,  635 

Wounds,  diphtheria  of,  dosage  of  anti- 
toxin in,  763 
infection,  antistreptococcus  serum  in, 

817 
vaccine,  therapy,  703 


Wright's  blood  capsules,  making  of,  23 
method  of  sealing,  34 
removing  serum  from,  34 
capillary  pipet  method  of  measuring 

bactericidal  power  of  blood,  377 
method  of  counting  bacterial  vaccines, 

YAWS,  luetin  reaction  in,  646 

salvarsan  in,  880 

Wassermann  reaction  in,  491 
Yersin's  plague  serum,  822 

ZOOPRECIPITIN,  314 
Zootoxins,  110,  121 

experimental  work,  899 


Nervous  and  Mental 
Diseases,   Children, 
Hygiene,   and    Medical 
Jurisprudence         l| 

W.  B.  SAUNDERS   COMPANY 

WEST  WASHINGTON  SQUARE  PHILADELPHIA 

9.  HENRIETTA  STREET        COVENT  GARDEN,  LONDON 

Dercum's   Mental   Diseases 

Clinical  Manual  of  Mental  Diseases.  By  FRANCIS  X.  DERCUM, 
PH.  D.,  M.  D.,  Professor  of  Nervous  and  Mental  Diseases  at  Jefferson 
Medical  College,  Philadelphia.  Octavo  ot  479  pages.  Cloth,  $3.50  net. 

SECOND  EDITION— Published  November,  1917 

This  is  a  book  really  useful  to  the  family  physician — a  book  that  tells  you 
definitely  how  to  diagnose,  how  to  treat — either  at  home  or  in  an  institution — all 
classes  of  mental  diseases.  First,  Dr.  Dercum  takes  up  the  various  primary 
forms  of  mental  disease,  giving  emphasis  to  those  you  meet  in  your  daily  practice 
as  genera.l  practitioner — delirium,  confusion,  stupor.  Then  melancholia,  mania, 
the  insanities  of  early  life,  paranoia,  the  neurasthenic-neuropathic  disorders,  and 
the  dementias  follow.  The  mental  disturbances  of  the  infections  (syphilis,  tubercu- 
losis, malaria,  pellagra,  rheumatic  fever,  etc.),  the  various  forms  of  intoxicational 
insanities,  those  due  to  metabolic  disorders,  visceral  disease,  diseases  of  the 
nervous  system  are  all  given  you — and  from  your  viewpoint.  An  important  sec- 
tion is  that  devoted  to  the  insanities  of  pregnancy.  An  entire  part  is  devoted  to 
the  psychologic  interpretations  of  symptoms  as  evolved  by  Freud  and  his  disciples. 
You  get  a  full  discussion  of  the  role  of  dreams. 

The  Medical  World 

'•  This  book  gives  just  the  information  necessary,  and  gives  it  in  a  style  studiously  adapted 
to  the  seeds  of  the  general  physician." 

Our  books  are  revised  frequently,  so  that  the 
editions  you  find  here  may  not  be  the  latest. 
Write  us  about  any  books  which  interest  you 


SAUNDERS'    BOOKS   ON 


Church  and  Peterson's 
Nervous  and  Mental  Diseases 


Nervous  and  Mental  Diseases.  By  ARCHIBALD  CHURCH,  M.  D., 
Professor  of  Nervous  and  Mental  Diseases  and  Medical  Jurisprudence, 
Northwestern  University  Medical  School,  Chicago;  and  FREDERICK 
PETERSON,  M.  D.,  formerly  Professor  of  Psychiatry  at  the  College  of 
Physicians  and  Surgeons,  N.  Y.  Handsome  octavo,  944  pages;  350 
illustrations.  Cloth,  $5.00  net.  October,  1914 

EIGHTH   EDITION 

For  this  new  (8th)  edition  this  standard  work  has  undergone  a  thorough  re- 
vision. Vertigo  and  its  labyrinthine  relations,  as  developed  by  Barany,  has 
received  careful  consideration ;  much  new  matter  has  been  added  to  the  section 
on  Infantile  Paralysis;  syphilis  of  the  nervous  system  has  been  brought  into  accord 
with  recent  epoch-making  discoveries.  Throughout,  references  to  the  new  inves- 
tigations of  the  spinal  fluid,  and  the  relation  of  spinal  fluid  changes  to  the  various 
organic  diseases  of  the  brain  and  cord  have  been  introduced.  The  bearing  of 
internal  secretion  upon  nervous  disorders  has  been  brought  right  down  to  date. 
Tetany  has  been  given  its  place  among  nervous  diseases  associated  with  glandular 
disorder.  Altogether  over  300  interpolations  and  alterations  have  been  made. 
It  is  more  than  ever  the.  standard. 

American  Journal  of  the  Medical  Sciences 

"  This  edition  has  been  revised,  new  illustrations  added,  and  some  new  matter,  and  really 
is  two  books.  .  .  .  The  descriptions  of  disease  are  clear,  directions  as  to  treatment  definite, 
and  disputed  matters  and  theories  are  omitted.  Altogether  it  is  a  most  useful  text-book." 


Kaplan's  Serology  of  Nervous  and 
Mental  Diseases 

Serology  of  Nervous  and  Mental  Diseases.  By  D.  M.  KAPLAN, 
M.  D.,  Director  of  Clinical  and  Research  Laboratories,  Neurological 
Institute,  New  York  City.  Octavo  of  346  pages,  illustrated.  Cloth, 

$3.5<D  net.  Published  June,  1914 


This  is  an  entirely  new  work,  giving  you  the  indications,  contra-indications, 
preparation  of  patients,  technic,  after-phenomena,  after-care,  and  disposal  of  the 
fluids  obtained  by  lumbar  puncture.  You  get  a  full  discussion  of  the  serology  of 
all  nervous. and  mental  diseases  of  non-luetic  etiology  (including  disorders  of 
internal  secretion),  and  of  every  type  of  luetic  nervous  and  mental  disease,  giv- 
ing the  Wassermann  reaction  in  detail,  the  use  of  salvarsan  and  neosaivarsan,  etc. 


MENTAL   DISEASES  AND  HYGIENE 


Herrick's  Neurology 

Introduction  to  Neurology.  By  C.  JUDSON  HERRICK,  PH.  D.,  Pro- 
fessor of  Neurology  in  the  University  of  Chicago.  1 2mo  of  360  pages, 

illustrated.       Cloth,  $1.75  net.  Published  September,  1 91 5 

A  KEY  TO  NEUROLOGY 

Professor  Herrick's  new  work  will  aid  the  student  to  organize  his  knowledge 
and  appreciate  the  significance  of  the  nervous  system  as  a  mechanism  right  at 
the  beginning  of  his  study.  It  is  sufficiently  elementary  to  be  used  by  students 
of  elementary  psychology  in  colleges  and  normal  schools,  by  students  of  general 
zoology  and  comparative  anatomy  in  college  classes,  and  by  medical  students  as 
a  guide  and  key  to  the  interpretation  of  the  larger  works  on  neurology. 

Herrick  and  Crosby's  Laboratory  Neurology 

A  Laboratory  Outline  of  Neurology.  By  C.  JUDSON  HERRICK, 
PH.  D.,  Professor  of  Neurology,  University  of  Chicago  ;  and  ELIZABETH 
C.  CROSBY,  PH.  D.,  Principal  of  the  High  School,  Petersburg,  Mich. 

I2mo  of  I2O  pages,  illustrated.       Cloth,  $I.5O  net.  Published  January,  1917 

Brill's  Psychanalysis  second  Edition  published  May,  19^ 

Psychanalysis :  Its  Theories  and  Practical  Application.  By  A.  A. 
BRILL,  PH.  B.,  M.  D.,  Clinical  Assistant  in  Neurology  at  Columbia 
University  Medical  School.  Octavo  of  392  pages.  Cloth,  $3.25  net. 

To  the  general  practitioner,  who  first  sees  these  "borderline"  cases  (the 
neuroses  and  the  psychoses),  as  well  as  to  those  specially  interested  in  neurologic 
work,  Dr.  Brill's  work  will  prove  most  valuable.  Dr.  Brill  has  had  wide  clinical 
experience,  both  in  America  and  in  Europe.  The  results  of  this  experience  you 
get  in  this  book.  Here  you  get  the  practical  application  of  all  Freud's  theories — 
and  from  the  pen  of  a  man  thoroughly  competent  to  write. 

Hunt's  Diagnostic  Symptoms  of  Nervous  Diseases 

Diagnostic  Symptoms  of  Nervous  Diseases.  By  EDWARD  L. 
HUNT,  M.  D.,  formerly  Instructor  in  Neurology  and  Assistant  Chief  of 
Clinic,  College  of  Physicians  and  Surgeons,  New  York.  I2mo  of  292 

pages,  illustrated.       Cloth,  $2.OO  net.  Second  Edition  published  June,  1917 

Stiles  on  the  Nervous  System 

The  Nervous  System  and  its  Conservation.  By  PERCY  G.  STILES, 
Instructor  in  Physiology  at  Harvard  University.  1 2mo  ot  240  pages, 

illustrated.       Cloth,  $.I5O  net.  Second  Edition  published  November,  1914 


SAUNDERS  BOOKS    ON 

• 

American  Illustrated  Dictionary 

New  (9th)  Edition— 2000  New  Words 

The  American  Illustrated  Medical  Dictionary.  A  new  and  com- 
plete dictionary  of  the  terms  used  in  Medicine,  Surgery,  Dentistry, 
Pharmacy,  Chemistry,  Veterinary  Science,  Nursing,  and  kindred 
branches;  with  over  100  new  and  elaborate  tables  and  many  hand- 
some illustrations.  By  W.  A.  NEWMAN  BORLAND,  M.  D.  Large 
octavo  1 179  pages,  bound  in  full  flexible  leather,  $5.00  net ;  with  thumb 

index,  $5.50  net.  Published  October,  1 91 7 

The  American  Illustrated  Medical  Dictionary  defines  hundreds  of  terms  not 
defined  in  any  other  dictionary — bar  none.  It  gives  the  capitalization  and  pro- 
nunciation of  all  words.  It  makes  a  feature  of  the  derivation  or  etymology  of  the 
words.  Every  word  has  a  separate  paragraph,  thus  making  it  easy  to  find  a 
word  quickly.  The  tables  of  arteries,  muscles,  nerves,  veins,  etc.,  are  of  the 
greatest  help  in  assembling  anatomic  facts.  Every  word  is  given  its  definition — a 
definition  that  defines  in  the  fewest  possible  words. 

Howard  A.  Kelly,  M.  D.,  Johns  Hopkins  University,  Baltimore. 

"  The  American  Illustrated  Dictionary  is  admirable.  It  is  so  well  gotten  up  and  of  such 
convenient  size.  No  errors  have  been  found  in  my  use  of  it." 

Owen's  Treatment  of  Emergencies 

The  Treatment  of  Emergencies.  By  HUBLEY  R.  OWEN,  M.  D., 
Surgeon  to  the  Philadelphia  General  Hospital.  I2mo  of  350  pages, 
with  249  illustrations.  Cloth,  $2.00  net.  Published  June,  191? 

Dr.  Owen's  book  is  a  complete  treatment  of  emergencies.  It  gives  you  not 
only  the  actual  technic  of  the  procedures,  but,  what  is  equally  important,  the  un- 
derlying principles  of  the  treatments,  and  the  reason  why  a  particular  method  is 
advised.  You  get  treatments  of  fractures,  of  contusions,  of  wounds.  Particularly 
strong  is  the  chapter  on  gun-shot  wounds,  which  gives  the  new  treatments  that  the 
great  European  War  has  developed.  You  get  the  principles  of  hemorrhage,  to- 
gether with  its  constitutional  and  local  treatments.  You  get  chapters  on  sprains, 
strains,  dislocations,  burns,  sunburn,  chilblain,  asphyxiation,  convulsions,  hysteria, 
apoplexy,  exhaustion,  opium  poisoning,  uremia,  electric  shock,  bandages,  and 
a  complete  discussion  of  artificial  respiration,  including  mechanical  devices. 


DISEASES   OF   CHILDREN. 


Kerley's  Pediatrics 

Practice  of  Pediatrics.  By  CHARLES  GILMORE  KERLEY,  *M.  D., 
Professor  of  Diseases  of  Children,  New  York  PolycKnic  Medical  School 
and  Hospital.  Octavo  of  913  pages,  illustrated.  Cloth,  $6.50  net. 

Published  January,  1918 
SECOND  EDITION 

This  work  is  not  a  cut-and-dried  treatise — but  the  practice  of  pediatrics,  giving 
fullest  attention  to  diagnosis  and  treatment.  The  chapters  on  the  newborn  and  its 
diseases,  the  feeding  and  growth  of  the  baby,  the  care  of  the  mother's  breasts, 
artificial  feeding,  milk  modification  and  sterilization,  diet  for  older  children — from 
a  monograph  of  125  pages.  Then  are  discussed  in  detail  every  disease  of  child- 
hood, telling  just  what  measures  should  be  instituted,  what  drugs  given,  60  valu- 
able prescriptions  being  included.  The  chapter  on  vaccine  therapy  is  right  down  to 
the  minute,  including  every  new  method  of  proved  value — with  the  exact  technic. 
There  is  an  excellent  chapter  on  Gymnastic  Therapeutics.  Another  feature  con- 
sists of  the  163  illustrative  cases — case  teaching  of  the  most  practical  sort. 

Dr.  A.  D.  Blackader,  Me  Gill  University,  Montreal 

"  Dr.  Kerley  is  a  pediatrician  of  large  experience  who  thinks  for  himself  and  is  n.eve-r  eon- 
tent  to  accept,  without  testing,  the  experiences  or  statements  of  previous  writers.  M4s  book  has 
a  very  definite  value." 

Bandler's  The  Expectant  Mother 

Published  August,  1916 

This  is  decidedly  a  book  for  the  woman  preparing  for  childbirth.  It  has 
chapters  on  menstruation,  nourishment  of  mother  during  pregnancy,  nausea, 
care  of  breasts,  examination  of  urine,  preparations  for  labor,  care  of  mother  and 
child  after  delivery,  twilight  sleep,  and  dozens  of  other  matters  of  great  interest 
to  the  expectant  mother. 

iamo  of  213  pages,  illustrated.  By  S.  WYLLIS  BANDLER,  M.  D.,  Professor  of  Diseases  of  Women,  New 
York  Post-Graduate  Medical  School  and  Hospital.  Cloth,  $1.25  net. 

Winslow's  Prevention  of  Disease 

Published  November,  1916 

This  book  is  a  practical  guide  for  the  layman,  giving  him  briefly  the  means 
to  avoid  the  various  diseases  described.  The  chapters  on  diet,  exercise,  tea, 
coffee,  and  alcohol  are  of  special  interest,  as  is  that  on  the  prevention*of  cancer. 
There  are  chapters  on  the  prevention  of  malaria,  colds,  constipation,  obesity, 
nervous  disorders,  tuberculosis,  etc.  The  work  is  a  record  of  twenty-five 
years'  active  practice. 

i2mo  of  348  pages,  illustrated.  By  KENELM  WINSLOW,  M.  D.,  formerly  Assistant  Professor  of  Com- 
parative Therapeutics,  Harvard  University.  Cloth,  $1.75  net. 

Kerr's  Diagnostics  of  Children's  Diseases 

Dr.  Kerr's  work  is  written  absolutely  for  the  general  practitioner — to  aid  him 
in  diagnosing  disease  in  his  child  patients.  He  approaches  his  subject  as  the 
child  is  approached  in  the  sick-room.  It  is  strictly  a  clinical  work — a  first  aid 
in  the  diagnosis  of  disease  in  children.  Published  February,  1907 

Octavo  of  542  pages,  illustrated.  By  LE  GRAND  KERR,  M.  D.,  Professor  of  Diseases  of  Children  in  the 
Brooklyn  Postgraduate  Medical  School.  Brooklyn.  Cloth,  $5.00  net. 


SAUNDERS'  BOOKS    ON 


Hill  and  Gerstley's  Infant  Feeding 

CLINICAL.  LECTURES  IN  INFANT  FEEDING.  By  LEWIS  WEBB  HILL, 
M.  D.,  Alumni  Assistant  in  Pediatrics,  Harvard  Medical  School,  and 
JESSE  R.  GERSTLEY,  M.  D.,  Instructor  in  Pediatrics,  Northwestern 
University  Medical  School.  I2mo  of  377  pages,  illustrated.  Cloth, 

$2.75  net.  Published  October,  1917 

In  these  clinics  you  are  given  the  full  details  of  the  Boston  method  of  infant 
feeding  as  developed  by  Dr.  Rotch,  and  of  the  Chicago  method.  You  are  given  the 
theory,  use  in  both  normal  and  abnormal  cases,  exact  quantities  and  percentages, 
and  concrete  clinical  examples.  The  book  is  equivalent  to  a  postgraduate  course 
in  infant  feeding.  It  brings  these  two  systems  right  to  your  door. 

Abt's  Preparation  of  Infants'  Foods 

THE  PREPARATION  OF  INFANTS'  FOODS.  By  ISAAC  A.  ABT,  M.D., 
Professor  of  Diseases  of  Children,  Northwestern  University  Medical 
School.  i2mo  of  143  pages.  Cloth,  $1.25  net.  Published  July,  1917 

This  is  a  practical  guide  for  infant  feeding,  giving  to  young  mothers,  nurses,  and 
caretakers  minute  directions  on  the  preparation  of  food  for  infants  and  young 
children.  You  get  weights  and  measures;  the  mineral  constituents  and  caloric 
values  of  foods.  You  get  such  practical  material  as  diet-lists  for  constipation  in 
older  children,  an  outline  of  a  plan  for  feeding  babies,  care  of  nipples  and  bottles, 
etc.,  and  a  great  host  of  recipes  for  beverages  of  all  kinds,  milk  preparations,  soups 
and  broths,  puddings  and  cereal  preparations,  custards,  eggs,  vegetables,  fruits, 
meats,  sea  foods,  and  breads. 


Aikens'  Home  Nurse's  Hand-Book 

HOME  NURSE'S  HAND-BOOK.  By  CHARLOTTE  A.  AIKENS.  i2mo  of 
303  pages,  illustrated.  Cloth,  $1.50  net. 

The  point  about  this  work  is  this  :  It  tells  you  and  shows  you  just  how  to  do  those 
little  but  important  things  often  omitted  from  other  nursing  books.  "Home  Treat- 
ments" and  "Points  to  be  Remembered"  —  terse,  crisp  reminders  —  stand  out  as  par- 
ticularly practical.  Just  the  book  for  those  who  have  the  home-care  of  the  sick. 


Galbraith's  Personal  Hygiene  for  Women 

PERSONAL  HYGIENE  AND  PHYSICAL  TRAINING  FOR  WOMEN.  By 
ANNA  M.  GALBRAITH,  M.  D.  i2mo  of  393  pages,  with  original  illus- 

trations.     Cloth,  $2.25  net.  Published  January,  1917 

"  It  contains  just  the  sort  of  information  which  is  very  greatly  needed  by  the  weaker 
sex.  Its  illustrations  are  excellent."  —  Dietetic  and  Hygienic  Gazette. 

Galbraith's  Four  Epochs  of  Woman's  Life  Ne£dit?odn 

THE  FOUR  EPOCHS  OF  WOMAN'S  LIFE.  By  ANNA  M.  GALBRAITH, 
M.D.  With  an  Introductory  Note  by  JOHN  H.  MUSSER,  M.,D.,  Uni- 
versity of  Pennsylvania.  i2mo  of  296  pages.  Cloth,  $1.50  net. 

"  We  do  not  as  a  rule  care  for  medical  books  written  for  the  instruction  of  the  public  ; 
but  we  must  admit  that  the  advice  in  Dr.  Galbraith's  work  is,  in  the  main,  wise  and  whole- 
some." —  Birmingham  Medical  Review.  Published  March,  1917 


CHILDREN  AND   HYGIENE 


Griffith's  Care  of  the  Baby 

The  Care  of  the  Baby.  By  J.  P.  CROZER  GRIFFITH,  M.  D.,  Professor 
of  Pediatrics  in  the  University  of  Pennsylvania.  I2mo  of  455  pages, 
illustrated.  '  Cloth,  $1.50  net. 

SIXTH  EDITION— published  June,  1915 

The  author  has  endeavored  to  furnish  a  reliable  guide  for  mothers.  He  has 
made  his  statements  plain  and  easily  understood,  so  that  the  volume  will  be  of 
service  to  mothers  and  nurses. 

New  York  Medical  Journal 

"We  are  confident  if  this  Itttle  work  could  find  its  n&f  into  the  hands  of  every  trained 
nurse  and  of  every  mother,  infant  mortality  would  be  lessened  by  at  least  fifty  per  cent." 


Grulee's  Infant  Feeding 

Infant  Feeding.  By  CLIFFORD  G.  GRULEE,  M.  D.,  Assistant  Pro- 
fessor of  Pediatrics  at  Rush  Medical  College.  Octavo  ot  326  pages,  illus- 
trated, including  8  in  colors.  Cloth,  $3.25  net. 

THIRD  EDITION— Publbhed  September,  1917 

Dr.  Grulee  tells  you  how  to  feed  the  infant.  He  tells  you — and  shows  by  clear 
illustrations — the  technic  of  giving  the  child  the  breast.  Then  artificial  feeding  is 
thoughtfully  presented,  including  a  number  of  simple  formulas.  The  colored  illus- 
trations showing  the  actual  shapes  and  appearances  of  stools  are  extremely 
valuable. 

Ruhrah's   Diseases   of    Children 

A  Manual  of  Diseases  of  Children.  By  JOHN  RUHRAH,  M.  D., 
Professor  of  Diseases  of  Children,  College  of  Physicians  and  Surgeons, 

Baltimore.     I2mo  of  552  pages,    fully    illustrated.     Flexible   leather, 
$2.75  net. 

FOURTH  EDITION— published  September,  1914 

In  revising  this  work  for  the  fourth  edition  Dr.  Ruhrah  has  carefully  in- 
corporated all  the  latest  knowledge  on  the  subject.  All  the  important  facts  are 
given  concisely  and  explicitly,  the  therapeutics  of  infancy  and  childhood  being 
outlined  very  carefully  and  clearly.  There  are  also  directions  for  dosage  and 
prescribing,  and  many  useful  prescriptions  are  included. 

American  Journal  of  the  Medical  Sciences 

"Treatment  has  been  satisfactorily  covered,  being  quite  in  accord  with  the  best  teaching, 
yet  withal  broadly  general  and  free  from  stock  prescriptions." 


SAUNDERS*    BOOKS    ON 


Reefer's  Military  Hygiene 

Military  Hygiene  and  Sanitation.  By  LIEUT.-COL.  FRANK  R, 
KEEPER,  Professor  of  Military  Hygiene,  United  States  Military  Academy, 
West  Point.  I2mo  of  305  pages,  illustrated.  Cloth,  $1.50  net. 

Published  July,  1914 

This  is  a  concise,  though  complete  text-book  on  this  subject,  containing 
chapters  on  the  care  of  troops,  recruits  and  recruiting,  personal  hygiene,  physical 
training,  preventable  diseases,  clothing,  equipment,  water-supply,  foods  and  their 
preparation,  hygiene  and  sanitation  of  posts  and  barracks,  the  troopship,  hygiene 
and  sanitation  of  marches,  camps,  and  battlefields,  disposal  of  wastes,  tropical  and 
arctic  service,  venereal  diseases,  alcohol  and  other  narcotics,  and  a  glossary. 


Sergey's  Hygiene 

The  Principles  of  Hygiene :  A  Practical  Manual  for  Students, 
Physicians,  and  Health  Officers.  By  D.  H.  BERGEY,  A.  M.,  M.  D., 
Assistant  Professor  of  Bacteriology  in  the  University  of  Pennsylvania. 
Octavo  volume  of  543  pages,  illustrated.  Cloth,  $3,50  net. 

SIXTH  EDITION— Published  February,   1918 

This  book  is  intended  to  meet  the  needs  of  students  of  medicine  in  the 
acquirement  of  a  knowledge  of  those  principles  upon  which  modern  hygienic 
practises  are  based,  and  to  aid  physicians  and  health  officers  in  familiarizing 
themselves  with  the  advances  made  in  hygiene  and  sanitation  in  recent  years. 
This  fifth  edition  has  been  very  carefully  revised,  and  much  new  matter 
added,  so  as  to  include  the  most  recent  advancements. 

Buffalo  Medical  Journal        * 

•'  It  will  be  found  of  value  to  the  practitioner  of  medicine  and  the  practical  sanitarian  ;  and 
students  of  architecture,  who  need  to  consider  problems  of  heating,  lighting,  ventilation,  water 
supply,  and  sewage  disposal,  may  consult  it  with  profit." 

Pyle's  Personal  Hygiene  New  (?th)  Edition-August,  1917 

A  MANUAL  OF  PERSONAL  HYGIENE  :  Proper  Living  upon  a  Physiologic 
Basis.  By  Eminent  Specialists.  Edited  by  WALTER  L.  PYLE,  A.  M., 
M.  D.,  Assistant  Surgeon  to  Wills  Eye  Hospital,  Philadelphia.  Octavo 
volume  of  555  pages,  fully  illustrated.  Cloth,  $i.75  net. 

The  book  has  been  thoroughly  revised  for  this  new  edition,  and  a  new  chapter  on 
Food  Adulteration  by  DR.  HARVEY  W.  WILEY  added.  There  are  important  chapters 
on  Domestic  Hygiene  and  Home  Gymnastics,  Hydrotherapy,  Mechanotherapy,  and 
First  Aid  Measures. 

"  The  work  has  been  excellently  done,  there  is  no  undue  repetition,  and  the  writers 
have  succeeded  unusually  well  in  presenting  facts  of  practical  significance  based  on  sound 
knowledge."— Boston  Medical  and  Surgical  Journal. 


LEGAL   MEDICINE 


Bohm  and  Painter's  Massage 

Massage.  By  MAX  BOHM,  M.  D.,  of  Berlin,  Germany.  Edited,  with  an 
Introduction,  by  CHARLES  F.  PAINTER,  M,  D.,  Professor  of  Orthopedic  Sur- 
gery at  Tufts  College  Medical  School,  Boston.  Octavo  of  91  pages,  with  97 
practical  illustrations.  Published  June,  1913  Cloth,  $1.75  net. 

Golebiewski  and  Bailey's  Accident  Diseases 

Atlas  and  Epitome  of  Diseases  Caused  by  Accidents.      By   DR.  ED. 

GOLEBIEWSKI,  of  Berlin.  Edited,  with  additions,  by  PEARCE  BAILEY,  M.  D., 
Consulting  Neurologist  to  St.  Luke's  Hospital,  New  York.  With  71  colored 
illustrations  on  40  plates,  143  text  illustrations,  and  549  pages  of  text.  Cloth, 
$4.00  net.  In  Saunders'  Hand-  Atlas  Series.  Published  1901 

Hofmann   and   Peterson's   Legal   Medicine 

Atlas  of  Legal  Medicine.  By  DR.  E.  VON  HOFMANN,  of  Vienna. 
Edited  by  FREDERICK  PETERSON,  M.  D.,  Professor  of  Psychiatry  in  the 
College  of  Physicians  and  Surgeons,  New  York.  With  120  colored  figures 
and  193  half-tone  illustrations.  Cloth,  $3.50  net.  Published  April,  1898 


Jakob  and  Fisher's  Nervous  System 

Atlases 
Atlas  and   Epitome  of  the  Nervous  System  and  its  Diseases.      By 

PROFESSOR  DR.  CHR.  JAKOB,  of  Erlangen.  Edited,  with  additions,  by  ED- 
WARD D.  FISHER,  M.  D.,  University  and  Bellevue  Hospital  Medical  College. 
With  83  plates  and  copious  text.  Cloth,  $3.  50  net.  Published  IQOI 

Spear's  Nervous  Diseases  Published  November,  wi6 

A  Manual  of  Nervous  Diseases.  By  IRVING  J.  SPEAR,  M.  D,.  Professor 
of  Neurology  at  the  University  of  Maryland,  Baltimore.  1  2mo  of  660  pages* 
illustrated.  Cloth,  $3.00  net. 

This  is  a  comprehensive  digest,  supplying  the  means  to  a  clear  understanding  of 
neurology,  and  robbing  that  subject  of  much  of  its  difficulty.  You  are  given,  first, 
a  brief  description  of  the  practical  anatomy  and  physiology,  with  those  facts  and 
theories  that  bear  on  the  mechanism  of  organic  nervous  diseases.  Then  pathology 
is  given,  the  simpler  diseases  being  considered  first,  gradually  preparing  the  reader 
to  grasp  the  more  difficult  ones.  The  descriptions  are  clear  and  brief,  differential 
diagnoses  and  treatments  being  brought  out  very  definitely.  Only  the  most  recent 
accepted  facts  have  been  considered.  For  the  treatments  recommended,  no  special 
apparatus  is  required  beyond  a  galvanic  and  faradic  battery;  they  demand  no 
special  training,  and  they  are  easily  remembered. 


I0  SAUNDERS'    BOOKS   ON  CHILDREN 

American  Pocket  Dictionary  New  (ioth)  Edition 

AMERICAN  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A,  NEW- 
MAN DORLAND,  M.  D.,  Editor  "American  Illustrated  Medical  Dic- 
tionary." Containing  the  pronunciation  and  definition  of  the  principal 
words  used  in  medicine  and  kindred  sciences,  with  75  extensive  tables. 
With  707  pages.  Flexible  leather,  with  gold  edges,  $1.25  net;  with 
patent  thumb  index,  $1.50  net.  Published  October,  1917 

"I  can  recommend  it  to  our  students  without  reserve."— J.  H.  HOLLAND,  M.  D., 
Dean  of  the  Jefferson  Medical  College,  Philadelphia. 

Morrow's  Immediate  Care  of  Injured  Third  Edition 

IMMEDIATE  CARE  OF  THE  INJURED.  By  ALBERT  S.  MORROW,  M.  D., 
Clinical  Professor  of  Surgery  at  the  New  York  Polyclinic.  Octavo  of  356 
pages,  with  242  illustrations.  Cloth,  $2.75  net.  Published  November,  1917 

Dr.  Morrow's  book  on  emergency  procedures  is  written  in  a  definite  and  decisive  style, 
the  reader  being  told  just  what  to  do  in  every  emergency.  It  is  a  practical  book  for  every 
day  use,  and  the  large  number  of  excellent  illustrations  can  not  but  make  the  treatment  to 
be  pursued  in  any  case  clear  and  intelligible.  Physicians  and  nurses  will  find  it  indispensible. 

Shaw  on  Nervous  Diseases  and  Insanity        Fifth  Edition 

ESSENTIALS  OF  NERVOUS  DISEASES  AND  INSANITY:  Their  Symptoms 
and  Treatment.  A  Manual  for  Students  and  Practitioners.  By  the  late 
JOHN  C.  SHAW,  M.  D.,  Clinical  Professor  of  Diseases  of  the  Mind  and 
Nervous  System,  Long  Island  College  Hospital,  New  York.  i2mo  of 
204  pages,  illustrated.  Cloth,  $1.25  net.  In  Saunders*  Question- Com- 

pend  Series.  Published  October,  1913 

"  Clearly  and  intelligently  written ;  we  have  noted  few  inaccuracies  and  several  sug- 
gestive points.  Some  affections  unmentioned  in  many  of  the  large  text-books  are  noted.' 
— Boston  Medical  and  Surgical  Journal. 

Brady's  Personal  Health  published  September,  me 

PERSONAL  HEALTH:  A  Doctor  Book  for  Discriminating  People.  By 
WILLIAM  BRADY,  M.D.,  Elmira,  N.  Y.  i2mo  of  406  pages.  Cloth,  $1.50  net 

Hecker,  Trumpp,  and  Abt  on  Children 

ATLAS  AND  EPITOME  OF  DISEASES  OF  CHILDREN.  By  DR.  R.  HECKER 
and  DR.  J.  TRUMPP,  of  Munich.  Edited,  with  additions,  by  ISAAC  A. 
ABT,  M.D.,  Assistant  Professor  of  Diseases  of  Children,  Rush  Medical 
College,  Chicago.  With  48  colored  plates,  144  text-cuts,  and  453  pages 
of  text.  Cloth,  $5.00  net.  Published  April,  1007 

The  many  excellent  lithographic  plates  represent  cases  seen  in  the  authors'  clinics,  and 
have  been  selected  with  great  care,  keeping  constantly  in  mind  the  practical  needs  of  the 
general  practitioner.  These  beautiful  pictures  are  so  true  to  nature  that  their  study  is 
equivalent  to  actual  clinical  observation.  The  editor,  Dr.  Isaac  A.  Abt,  has  added  all  new 
methods  of  treatment. 


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URRAUT 


